Introduction

Gas chromatography - specifically gas-liquid chromatography - involves a sample being vapourised and injected onto the head of the chromatographic column. The sample is transported through the column by the flow of inert, gaseous mobile phase. The column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid. Have a look at this schematic diagram of a gas chromatograph:

Instrumental components
Carrier gas The carrier gas must be chemically inert. Commonly used gases include nitrogen, helium, argon, and carbon dioxide. The choice of carrier gas is often dependant upon the type of detector which is used. The carrier gas system also contains a molecular sieve to remove water and other impurities.

Sample injection port For optimum column efficiency, the sample should not be too large, and should be introduced onto the column as a "plug" of vapour - slow injection of large samples causes band broadening and loss of resolution. The most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port

at the head of the column. The temperature of the sample port is usually about 50C higher than the boiling point of the least volatile component of the sample. For packed columns, sample size ranges from tenths of a microliter up to 20 microliters. Capillary columns, on the other hand, need much less sample, typically around 10-3 L. For capillary GC, split/splitless injection is used. Have a look at this diagram of a split/splitless injector;

The injector can be used in one of two modes; split or splitless. The injector contains a heated chamber containing a glass liner into which the sample is injected through the septum. The carrier gas enters the chamber and can leave by three routes (when the injector is in split mode). The sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes. A proportion of this mixture passes onto the column, but most exits through the split outlet. The septum purge outlet prevents septum bleed components from entering the column.

Columns There are two general types of column, packed and capillary (also known as open tubular). Packed columns contain a finely divided, inert, solid support material (commonly based on diatomaceous earth) coated with liquid stationary phase. Most packed columns are 1.5 - 10m in length and have an internal diameter of 2 - 4mm. Capillary columns have an internal diameter of a few tenths of a millimeter. They can be one of two types; wall-coated open tubular (WCOT) or support-coated open tubular

(SCOT). Wall-coated columns consist of a capillary tube whose walls are coated with liquid stationary phase. In support-coated columns, the inner wall of the capillary is lined with a thin layer of support material such as diatomaceous earth, onto which the stationary phase has been adsorbed. SCOT columns are generally less efficient than WCOT columns. Both types of capillary column are more efficient than packed columns. In 1979, a new type of WCOT column was devised - the Fused Silica Open Tubular (FSOT) column;

These have much thinner walls than the glass capillary columns, and are given strength by the polyimide coating. These columns are flexible and can be wound into coils. They have the advantages of physical strength, flexibility and low reactivity.

Column temperature For precise work, column temperature must be controlled to within tenths of a degree. The optimum column temperature is dependant upon the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 - 30 minutes. Minimal temperatures give good resolution, but increase elution times. If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.

Detectors There are many detectors which can be used in gas chromatography. Different detectors will give different types of selectivity. A non-selective detector responds to all compounds except the carrier gas, a selective detector responds to a range of compounds with a common physical or chemical property and a specific detector responds to a single chemical compound. Detectors can also be grouped into concentration dependant detectors and mass flow dependant detectors. The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample Dilution of with make-up gas will lower the detectors response. Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate

at which solute molecules enter the detector. The response of a mass flow dependant detector is unaffected by make-up gas. Have a look at this tabular summary of common GC detectors: Detector Type Support gases Selectivity Most organic cpds. Detectability 100 pg Dynamic range 107

Flame Hydrogen ionization Mass flow and air (FID) Thermal conductivity Concentration Reference (TCD) Electron capture (ECD) Nitrogenphosphorus Flame photometric (FPD) Concentration Make-up Hydrogen and air Hydrogen and air possibly oxygen

Universal Halides, nitrates, nitriles, peroxides, anhydrides, organometallics

1 ng

107

50 fg

105

Mass flow

Nitrogen, phosphorus 10 pg Sulphur, phosphorus, tin, boron, arsenic, 100 pg germanium, selenium, chromium Aliphatics, aromatics, ketones, esters, aldehydes, amines, 2 pg heterocyclics, organosulphurs, some organometallics

106

Mass flow

103

Photoionization (PID)

Concentration Make-up

107

Hall electrolytic Mass flow conductivity

Hydrogen, Halide, nitrogen, oxygen nitrosamine, sulphur

The effluent from the column is mixed with hydrogen and air, and ignited. Organic compounds burning in the flame produce ions and electrons which can conduct electricity through the flame. A large electrical potential is applied at the burner tip, and a collector electrode is located above the flame. The current resulting from the pyrolysis of any organic compounds is measured. FIDs are mass sensitive rather than concentration sensitive; this gives the advantage that changes in mobile phase flow rate do not affect the detector's response. The FID is a useful general detector for the analysis of organic compounds; it has high sensitivity, a large linear response range, and low noise. It is also robust and easy to use, but unfortunately, it destroys the sample.

Introductory theory
Introduction
Few methods of chemical analysis are truly specific to a particular analyte. It is often found that the analyte of interest must be separated from the myriad of individual compounds that may be present in a sample. As well as providing the analytical scientist with methods of separation, chromatographic techniques can also provide methods of analysis.

Chromatography involves a sample (or sample extract) being dissolved in a mobile phase (which may be a gas, a liquid or a supercritical fluid). The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase. Techniques such as H.P.L.C. (High Performance Liquid Chromatography) and G.C. (Gas Chromatography) use columns - narrow tubes packed with stationary phase, through which the mobile phase is forced. The sample is transported through the column by continuous addition of mobile phase. This process is called elution. The average rate at which an analyte moves through the column is determined by the time it spends in the mobile phase.

Distribution of analytes between phases

The distribution of analytes between phases can often be described quite simply. An analyte is in equilibrium between the two phases;

Amobile

Astationary

The equilibrium constant, K, is termed the partition coefficient; defined as the molar concentration of analyte in the stationary phase divided by the molar concentration of the analyte in the mobile phase. The time between sample injection and an analyte peak reaching a detector at the end of the column is termed the retention time (tR ). Each analyte in a sample will have a different retention time. The time taken for the mobile phase to pass through the column is called tM.

A term called the retention factor, k', is often used to describe the migration rate of an analyte on a column. You may also find it called the capacity factor. The retention factor for analyte A is defined as;

k'A = t R - tM / tM
t R and tM are easily obtained from a chromatogram. When an analytes retention factor is less than one, elution is so fast that accurate determination of the retention time is very difficult. High retention factors (greater than 20) mean that elution takes a very long time. Ideally, the retention factor for an analyte is between one and five. We define a quantity called the selectivity factor, , which describes the separation of two species (A and B) on the column;

= k 'B / k 'A
When calculating the selectivity factor, species A elutes faster than species B. The selectivity factor is always greater than one.

Band broadening and column efficiency

To obtain optimal separations, sharp, symmetrical chromatographic peaks must be obtained. This means that band broadening must be limited. It is also beneficial to measure the efficiency of the column.

The Theoretical Plate Model of Chromatography The plate model supposes that the chromatographic column is contains a large number of separate layers, called theoretical plates. Separate equilibrations of the sample between the stationary and mobile phase occur in these "plates". The analyte moves down the column by transfer of equilibrated mobile phase from one plate to the next.

It is important to remember that the plates do not really exist; they are a figment of the imagination that helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better). If the length of the column is L, then the HETP is

HETP = L / N The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution;

where w1/2 is the peak width at half-height. As can be seen from this equation, columns behave as if they have different numbers of plates for different solutes in a mixture.

The Rate Theory of Chromatography A more realistic description of the processes at work inside a column takes account of the time taken for the solute to equilibrate between the stationary and mobile phase (unlike the plate model, which assumes that equilibration is infinitely fast). The resulting band shape of a chromatographic peak is therefore affected by the rate of elution. It is also affected by the different paths available to solute molecules as they travel between particles of stationary phase. If we consider the various mechanisms which contribute to band broadening, we arrive at the Van Deemter equation for plate height; HETP = A + B / u + C u where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening. A - Eddy diffusion The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths. B - Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion. C - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of

the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes. Van Deemter plots A plot of plate height vs. average linear velocity of mobile phase.

Such plots are of considerable use in determining the optimum mobile phase flow rate.

Resolution
Although the selectivity factor, , describes the separation of band centres, it does not take into account peak widths. Another measure of how well species have been separated is provided by measurement of the resolution. The resolution of two species, A and B, is defined as

Baseline resolution is achieved when R = 1.5 It is useful to relate the resolution to the number of plates in the column, the selectivity factor and the retention factors of the two solutes;

To obtain high resolution, the three terms must be maximised. An increase in N, the number of theoretical plates, by lengthening the column leads to an increase in retention time and increased band broadening - which may not be desirable. Instead, to increase the

number of plates, the height equivalent to a theoretical plate can be reduced by reducing the size of the stationary phase particles. It is often found that by controlling the capacity factor, k', separations can be greatly improved. This can be achieved by changing the temperature (in Gas Chromatography) or the composition of the mobile phase (in Liquid Chromatography). The selectivity factor, , can also be manipulated to improve separations. When is close to unity, optimising k' and increasing N is not sufficient to give good separation in a reasonable time. In these cases, k' is optimised first, and then is increased by one of the following procedures: 1. 2. 3. 4. Changing mobile phase composition Changing column temperature Changing composition of stationary phase Using special chemical effects (such as incorporating a species which complexes with one of the solutes into the stationary phase)

Gas Chromatography
This technique was first carried out in Austria, and the first exploitation of the method was made by Martin and A T James in 1952, when they reported the gas chromatography of organic acids and amines. The "support" was coated with a non-volatile liquid and placed into a heated glass tube. Mixtures injected into the tube and carried through by compressed gas resulted in well defined zones. This development was a great asset to petroleum chemists who recognized it as a simple and rapid method of analysis of the complex hydrocarbon mixtures encountered in petroleum products. A major advance came with the elimination of the support material and the coating of the liquid onto the wall of a long capillary tube; the advent of capillary columns. This development made it possible to carry out separations of many different components in a single chromatographic analysis. The discovery of the structure of insulin, for example, was made possible when the British biochemist, Frederick Sanger, rationally and methodically applied the technique to the fragments of the ruptured insulin molecule, for which he received the 1958 Nobel prize for chemistry.(1)

THE PRINCIPLES OF GAS CHROMATOGRAPHY

Chromatography (2) is the separation of a mixture of compounds (solutes) into separate components. By separating the sample into individual components, it is easier to identify (qualitate) and measure the amount (quantitate) of the various sample components. There are numerous chromatographic techniques and corresponding instruments. Gas chromatography (GC) is one of these techniques. It is estimated that 10-20% of the known compounds can be analyzed by GC. To be suitable for GC analysis, a compound must have sufficient volatility and thermal stability. If all or some of a compound or molecules are in the gas or vapor phase at 400450C or below, and they do not decompose at these temperatures, the compound can probably be analyzed by GC. One or more high purity gases are supplied to the GC. One of the gases (called the carrier gas)

flows into the injector, through the column and then into the detector. A sample is introduced into the injector usually with a syringe or an exterior sampling device. The injector is usually heated to 150-250C which causes the volatile sample solutes to vaporize. The vaporized solutes are transported into the column by the carrier gas. The column is maintained in a temperature controlled oven. The solutes travel through the column at a rate primarily determined by their physical properties, and the temperature and composition of the column. The various solutes travel through the column at different rates. The fastest moving solute exits (elutes) the column first then is followed by the remaining solutes in corresponding order. As each solute elutes from the column, it enters the heated detector. An electronic signal is generated upon interaction of the solute with the detector. The size of the signal is recorded by a data system and is plotted against elapsed time to produce a chromatogram. The ideal chromatogram has closely spaced peaks with no overlap of the peaks. Any peaks that overlap are called coeluting. The time and size of a peak are important in that they are used to identify and measure the amount of the compound in the sample. The size of the resulting peak corresponds to the amount of the compound in the sample. A larger peak is obtained as the concentration of the corresponding compound increases. If the column and all of operating conditions are kept the same, a given compound always travels through the column at the same rate. Thus, a compound can be identified by the time required for it to travel through the column (called the retention time). The identity of a compound cannot be determined solely by its retention time. A known amount of an authentic, pure sample of the compound has to be analyzed and its retention time and peak size determined. This value can be compared to the results from an unknown sample to determine whether the target compound is present (by comparing retention times) and its amount (by comparing peak sizes). If any of the peaks overlap, accurate measurement of these peaks is not possible. If two peaks have the same retention time, accurate identification is not possible. Thus, it is desirable to have no peak overlap or co-elution

RETENTION TIME (tR)

Retention time (tR) is the time it takes a solute to travel through the column. The retention time is assigned to the corresponding solute peak. The retention time is a measure of the amount of time a solute spends in a column. It is the sum of the time spent in the stationary phase and the mobile phase.

COLUMN BLEED:
Column bleed is the background generated by all columns. It is the continuous elution of the compounds produced from normal degradation of the stationary phase. Column bleed increases at higher temperatures.

COLUMN TEMPERATURE LIMITS:

Columns have lower and upper temperature limits. If a column is used below its lower temperature limit, rounded and wide peaks are obtained (i.e., loss of efficiency). No column damage has occurred; however, the column does not function properly. Using the column at or above its lower limit maintains good peak shapes. Upper temperature limits are often stated as two numbers. The lower one is the isothermal temperature limit. The column can be used indefinitely at this temperature and reasonable column bleed and lifetime are realized. The upper number is the temperature program limit. A column can be maintained at this temperature for 10-15 minutes without severely shortening column lifetime or experiencing

excessively high column bleed. Exposing the column to higher temperatures or for longer time periods results in higher column bleed and shorter column lifetimes. Exceeding the upper temperature limits may damage the stationary phase and the inertness of the fused silica tubing.

COLUMN CAPACITY:
Column capacity is the maximum amount of a solute that can be introduced into a column before significant peak distortion occurs. Overloaded peaks are asymmetric with a leading edge. Overloaded peaks are often described as "shark fin" shaped. Tailing peaks are obtained if a PLOT column is overloaded. No damage occurs if a column is overloaded Back to top

STATIONARY PHASES

POLYSILOXANES:
Polysiloxanes are the most common stationary phases. They are available in the greatest variety and are the most stable, robust and versatile. The most basic polysiloxane is the 100% methyl substituted. When other groups are present, the amount is indicated as the percent of the total number of groups. For example, a 5% diphenyl95% dimethyl polysiloxane contains 5% phenyl groups and 95% methyl groups. The "di-" prefix indicates that each silicon atom contains two of that particular group. Sometimes this prefix is omitted even though two identical groups are present. If the methyl percentage is not stated, it is understood to be present in the amount necessary to make 100% (e.g., 50% phenyl-methyl polysiloxane contains 50% methyl substitution). Cyanopropylphenyl percent values can be misleading. A 14% cyanopropylphenyl-dimethyl polysiloxane contains 7% cyanopropyl and 7% phenyl (along with 86% methyl). The cyanopropyl and phenyl groups are on the same silicon atom, thus their amounts are summed.

POLYETHYLENE GLYCOLS:
Polyethylene glycols (PEG) are widely used as stationary phases. Stationary phases with "wax" or "FFAP" in their name are some type of polyethylene glycol. Polyethylene glycols stationary phases are not substituted, thus the polymer is 100% of the stated material. They are less stable, less robust and have lower temperature limits than most polysiloxanes. With typical use, they exhibit shorter lifetimes and are more susceptible to damage upon over heating or exposure to oxygen. The unique separation properties of polyethylene glycol makes these liabilities tolerable. Polyethylene glycol stationary phases must be liquids under GC temperature conditions.

GAS - SOLID:(PLOT Columns)

Gas-solid stationary phases are comprised of a thin layer (usually <10 m) of small particles adhered to the surface of the tubing. These are porous layer open tubular (PLOT) columns. The sample compounds undergo a gassolid adsorption/desorption process with the stationary phase. The particles are porous, thus size

exclusion and shape selectivity processes also occur. Various derivatives of styrene, aluminum oxides and molecular sieves are the most common PLOT column stationary phases. PLOT columns are very retentive. They are used to obtain separations that are impossible with conventional stationary phases. Also, many separations requiring subambient temperatures with polysiloxanes or polyethylene glycols can be easily accomplished above ambient temperatures with PLOT columns. Hydrocarbon and sulfur gases, noble and permanent gases, and low boiling point solvents are some of the more common compounds separated with PLOT columns. Some PLOT columns may occasionally lose particles of the stationary phase. For this reason, using PLOT columns that may lose particles with detectors negatively affected by particulate matter is not recommended. Mass spectrometers are particularly susceptible to this problem due to the presence of a strong vacuum at the exit of the column.

BONDED AND CROSS-LINKED STATIONARY PHASES:

Cross-linked stationary phases have the individual polymer chains linked via covalent bonds. Bonded stationary phases are covalently bonded to the surface of the tubing. Both techniques impart enhanced thermal and solvent stability to the stationary phase. Also, columns with bonded and cross-linked stationary phases can be solvent rinsed to remove contaminants. Most polysiloxanes and polyethylene glycol stationary phases are bonded and cross-linked. A few stationary phases are available in an nonbonded version; some stationary phases are not available in bonded and cross-linked versions. Use a bonded and cross-linked stationary phase if one is available. Back to top

GAS CHROMATOGRAPHY DETECTION

DETECTORS
As solutes elute from the column, they interact with the detector. The detector converts this interaction into an electronic signal that is sent to the data system. The magnitude of the signal is plotted versus time (from the time of injection) and a chromatogram is generated. Some detectors respond to any solute eluting from the column while others respond only to solutes with specific structures, functional groups or atoms Detectors that exhibit enhanced response to specific types of solutes are called selective detectors. Most detectors require one or more gases to function properly. There are combustion, reagent, auxiliary and makeup gases. In some cases, one gas may serve multiple purposes. The type of detector gas is dependent on the specific detector and is fairly universal between GC manufacturers. The flow rates for each type of detector varies between GC manufacturers. It is important to follow the recommended flow rates to obtain the optimal sensitivity, selectivity and linear range for a detector

FLAME IONIZATION DETECTOR (FID):

Mechanism: Compounds are burned in a hydrogen-air flame. Carbon containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Selectivity: Compounds with C-H bonds. A poor response for some non-hydrogen containing organics (e.g., hexachlorobenzene). Sensitivity: 0.1-10 ng Linear range: 105-107 Gases: Combustion - hydrogen and air; Makeup - helium or nitrogen Temperature: 250-300C,and 400-450C for high temperature analyses.

NITROGEN PHOSPHORUS DETECTOR (NPD):

Mechanism: Compounds are burned in a plasma surrounding a rubidium bead supplied with hydrogen and air. Nitrogen and phosphorous containing compounds produce ions that are attracted to the collector. The number of ions hitting the collector is measured and a signal is generated. Selectivity: Nitrogen and phosphorous containing compounds Sensitivity: 1-10 pg Linear range: 104-10-6 Gases: Combustion - hydrogen and air; Makeup - helium Temperature: 250-300C

ELECTRON CAPTURE DETECTOR (ECD):

Mechanism: Electrons are supplied from a 63Ni foil lining the detector cell. A current is generated in the cell. Electronegative compounds capture electrons resulting in a reduction in the current. The amount of current loss is indirectly measured and a signal is generated. Selectivity: Halogens, nitrates and conjugated carbonyls Sensitivity: 0.1-10 pg (halogenated compounds); 1-100 pg (nitrates); 0.1-1 ng (carbonyls) Linear range: 103-104 Gases: Nitrogen or argon/methane Temperature: 300-400C

THERMAL CONDUCTIVITY DETECTOR (TCD):

Mechanism: A detector cell contains a heated filament with an applied current. As carrier gas containing solutes passes through the cell, a change in the filament current occurs. The current change is compared against the current in a reference cell. The difference is measured and a signal is generated. Selectivity: All compounds except for the carrier gas Sensitivity: 5-20 ng Linear range: 105-106 Gases: Makeup - same as the carrier gas Temperature: 150-250C

FLAME PHOTOMETRIC DETECTOR (FPD):

Mechanism: Compounds are burned in a hydrogen-air flame. Sulfur and phosphorous containing compounds produce light emitting species (sulfur at 394 nm and phosphorous at 526 nm). A

monochromatic filter allows only one of the wavelengths to pass. A photomultiplier tube is used to measure the amount of light and a signal is generated. A different filter is required for each detection mode. Selectivity: Sulfur or phosphorous containing compounds. Only one at a time. Sensitivity: 10-100 pg (sulfur); 1-10 pg (phosphorous) Linear range: Non-linear (sulfur); 103-105 (phosphorous) Gases: Combustion - hydrogen and air; Makeup - nitrogen Temperature: 250-300C

PHOTOIONIZATION DETECTOR (PID):

Mechanism: Compounds eluting into a cell are bombarded with high energy photons emitted from a lamp. Compounds with ionization potentials below the photon energy are ionized. The resulting ions are attracted to an electrode, measured, and a signal is generated. Selectivity: Depends on lamp energy. Usually used for aromatics and olefins (10 eV lamp). Sensitivity: 25-50 pg (aromatics); 50-200 pg (olefins) Linear range: 105-106 Gases: Makeup - same as the carrier gas Temperature: 200C

ELECTROLYTIC CONDUCTIVITY DETECTOR (ELCD):

Mechanism: Compounds are mixed with a reaction gas and passed through a high temperature reaction tube. Specific reaction products are created which mix with a solvent and pass through an electrolytic conductivity cell. The change in the electrolytic conductivity of the solvent is measured and a signal is generated. Reaction tube temperature and solvent determine which types of compounds are detected. Selectivity: Halogens, sulfur or nitrogen containing compounds. Only one at a time. Sensitivity: 5-10 pg (halogens); 10-20 pg (S); 10-20 pg (N) Linear range: 105-106 (halogens); 104-105 (N); 103.5-104(S) Gases: Hydrogen (halogens and nitrogen); air (sulfur) Temperature: 800-1000C (halogens), 850-925C (N), 750-825C (S)

MASS SPECTROMETER (MS):

Mechanism: The detector is maintained under vacuum. Compounds are bombarded with electrons (EI) or gas molecules (CI). Compounds fragment into characteristic charged ions or fragments. The resulting ions are focused and accelerated into a mass filter. The mass filter selectively allows all ions of a specific mass to pass through to the electron multiplier. All of the ions of the specific mass are detected. The mass filter then allows the next mass to pass through while excluding all others. The mass filter scans stepwise through the designated range of masses several times per second. The total number of ions are counted for each scan. The abundance or number of ions per scan is plotted versus time to obtain the chromatogram (called the TIC). A mass spectrum is obtained for each scan which plots the various ion masses versus their abundance or number. Selectivity: Any compound that produces fragments within the selected mass range. May be an inclusive range of masses (full scan) or only select ions (SIM). Sensitivity: 1-10 ng (full scan); 1-10 pg (SIM) Linear range: 105-106 Gases: None Temperature: 250-300C (transfer line), 150-250C (source) Back to top

COLUMN DEGRADATION

COLUMN BREAKAGE:
Fused silica columns break wherever there is a weak point in the polyimide coating. The polyimide coating protects the fragile fused silica tubing. The continuous heating and cooling of the oven, vibrations caused by the oven fan and being wound on a circular cage all place stress on the tubing. Eventually breakage occurs at a weak point. Weak spots are created when the polyimide coating is scratched or abraded. This usually occurs when a sharp point or edge is dragged over the tubing. Column hangers and tags, metal edges in the GC oven, column cutters and miscellaneous items on the lab bench are just some of the common sources of sharp edges or points. It is rare for a column to spontaneously break. Column manufacturing practices tend to expose any weak tubing and eliminate it from use in finished columns. Larger diameter columns are more prone to breakage. This means that greater care and prevention against breakage must be taken with 0.45-0.53 mm I.D. tubing than with 0.18-0.32 mm I.D. tubing. A broken column is not always fatal. If a broken column was maintained at a high temperature either continuously or with multiple temperature program runs, damage to the column is very likely. The back half of the broken column has been exposed to oxygen at elevated temperatures which rapidly damages the stationary phase The front half is fine since carrier gas flowed through this length of column. If a broken column has not been heated or only exposed to high temperatures or oxygen for a very short time, the back half has probably not suffered any significant damage. A union can be installed to repair a broken column. Any suitable union will work to rejoin the column. No more than 2-3 unions should be installed on any one column. Problems with dead volume (peak tailing) may occur with multiple unions.

THERMAL DAMAGE:
Exceeding a column upper temperature limit results in accelerated degradation of the stationary phase and tubing surface. This results in the premature onset of excessive column bleed, peak tailing for active compounds and/or loss of efficiency (resolution). Fortunately, thermal damage is a slower process, thus prolonged times above the temperature limit are required before significant damage occurs. Thermal damage is greatly accelerated in the presence of oxygen. Overheating a column with a leak or high oxygen levels in the carrier gas results in rapid and permanent column damage. Setting the maximum oven temperature at or a few degrees above the column temperature limit is the best method to prevent thermal damage. This prevents the accidental overheating of the column. If a column is thermally damaged, it may still be functional. Remove the column from the detector. Heat the column for 8-16 hours at its isothermal temperature limit. Remove 10-15 cm from the detector end of the column. Reinstall the column and condition as usual. The column usually does not return to its original performance; however, it is often still functional. The life of the column will be reduced after thermal damage.

OXYGEN DAMAGE:
Oxygen is an enemy to most capillary GC columns. While no column damage occurs at or near ambient temperatures, severe damage occurs as the column temperature increases. In general, the temperature and oxygen concentration at which significant damages occurs is lower for polar stationary phases. It is constant exposure to oxygen that is the problem. Momentary exposure such as an injection of air or a very short duration septum nut removal is not a problem. A leak in the carrier gas flow path (e.g., gas lines, fittings, injector) is the most common source of oxygen exposure. As the column is heated, very rapid degradation of the stationary phase occurs.

This results in the premature onset of excessive column bleed, peak tailing for active compounds and/or loss of efficiency (resolution). These are the same symptoms as for thermal damage. Unfortunately, by the time oxygen damage is discovered, significant column damage has already occurred. In less severe cases, the column may still be functional but at a reduced performance level. In more severe cases, the column is irreversibly damaged. Maintaining an oxygen and leak free system is the best prevention against oxygen damage. Good GC system maintenance includes periodic leak checks of the gas lines and regulators, regular septa changes, using high quality carrier gases, installing and changing oxygen traps, and changing gas cylinders before they are completely empty.

CHEMICAL DAMAGE:
There are relatively few compounds that damage stationary phases. Introducing non-volatile compounds (high molecular weight or high boiling point) in a column often degrades performance, but damage to the stationary phase does not occur. These residues can often be removed and performance returned by solvent rinsing the column Inorganic or mineral bases and acids are the primary compounds to avoid introducing in a column. The acids include hydrochloric (HCl), sulfuric (H2SO4), nitric (HNO3), phosphoric (H3PO4) and chromic (CrO3). The bases include potassium hydroxide (KOH), sodium hydroxide (NaOH) and ammonium hydroxide (NH4OH). Most of these acids and bases are not very volatile and accumulate at the front of the column. If allowed to remain, the acids or bases damage the stationary phase. This results in the premature onset of excessive column bleed, peak tailing for active compounds and/or loss of efficiency (resolution). The symptoms are very similar to thermal and oxygen damage. Hydrochloric acid and ammonium hydroxide are the least harmful of the group. Both tend to follow any water that is present in the sample. If the water is not or only poorly retained by the column, the residence time of HCl and NH4OH in the column is short. This tends to eliminate or minimize any damage by these compounds. Thus, if HCl or NH4OH are present in a sample, using conditions or a column with no water retention will render these compounds relatively harmless to the column. The only organic compounds that have been reported to damage stationary phases are perfluoroacids. Examples include trifluoroacetic, pentafluoropropanoic and heptafluorobutyric acid. They need to be present at high levels (e.g., 1% or higher). Most of the problems are experienced with splitless or Megabore direct injections where large volumes of the sample are deposited at the front of the column. Since chemical damage is usually limited to the front of the column, trimming or cutting 1/2-1 meter from the front of the column often eliminates any chromatographic problems. In more severe cases, 5 or more meters may need to be removed. The use of a guard column or retention gap will minimize the amount of column damage; however, frequent trimming of the guard column may be necessary. The acid or base often damages the surface of the deactivated fused silica tubing which leads to peak shape problems for active compounds.

COLUMN CONTAMINATION
Column contamination is one of the most common problems encountered in capillary GC. Unfortunately, it mimics a very wide variety of problems and is often misdiagnosed as another problem. A contaminated column is usually not damaged, but it may be rendered unusable. There are two basic types of contaminants: nonvolatile and semi-volatile. Nonvolatile contaminants or residues do not elute and accumulate in the column. The column becomes coated with these residues which interfere with the proper partitioning of solutes in and out of the stationary phase. Also, the residues may interact with active solutes resulting in peak adsorption problems (evident as peak tailing or loss of peak size). Active solutes are those containing a

hydroxyl (-OH) or amine (-NH) group, and some thiols (-SH) and aldehydes. Semivolatile contaminants or residues accumulate in the column, but eventually elute. Hours to days may elapse before they completely leave the column. Like nonvolatile residues, they may cause peak shape and size problems and, in addition, are usually responsible for many baseline problems (instability, wander, drift, ghost peaks, etc.). Contaminants originate from a number of sources with injected samples being the most common. Extracted samples are among the worse types. Biological fluids and tissues, soils, waste and ground water, and similar types of matrices contain high amounts of semivolatile and nonvolatile materials. Even with careful and thorough extraction procedures, small amounts of these materials are present in the injected sample. Several to hundreds of injections may be necessary before the accumulated residues cause problems. Injection techniques such as on-column, splitless and Megabore direct place a large amount of sample into the column, thus column contamination is more common wirh these injection techniques. Occasionally contaminants originate from materials in gas lines and traps, ferrule and septa particles, or anything coming in contact with the sample (vials, solvents, syringes, pipettes, etc.). These types of contaminants are probably responsible when a contamination problem suddenly develops and similar samples in previous months or years did not cause any problems. Minimizing the amount of semivolatiles and nonvolatile sample residues is the best method to reduce contamination problems. Unfortunately, the presence and identity of potential contaminants are often unknown. Rigorous and thorough sample cleanup is the best protection against contamination problems. The use of a guard column or retention gap often reduces the severity or delays the onset of column contamination induced problems. If a column becomes contaminated, it is best to solvent rinse the column to remove the contaminants. Maintaining a contaminated column at high temperatures for long periods of time (often called baking out a column) is not recommended. Baking out a column may convert some of the contaminating residues into insoluble materials that cannot be solvent rinsed from the column. If this occurs, the column cannot be salvaged in most cases. Sometimes the column can be cut in half and the back half may still be useable. Baking out a column should be limited to 1-2 hours at the isothermal temperature limit of the column. Back to top

PROBLEMS IN GAS CHROMATOGRAPHY TROUBLESHOOTING

EVALUATING THE PROBLEM:

The first step in any troubleshooting effort is to step back and evaluate the situation. Rushing to solve the problem often results in a critical piece of important information being overlooked or neglected. In addition to the problem, look for any other changes or differences in the chromatogram. Many problems are accompanied by other symptoms. Retention time shifts, altered baseline noise or drift, or peak shape changes are only a few of the other clues that often point to or narrow the list of possible causes. Finally, make note of any changes or differences involving the sample. Solvents, vials, pipettes, storage conditions, sample age, extraction or preparation techniques, or any other factor influencing the sample environment can be responsible.

SIMPLE CHECKS AND OBSERVATIONS:

A surprising number of problems involve fairly simple and often overlooked components of the GC system or analysis. Many of these items are transparent in the daily operation of the GC and are often taken for granted (set it and forget it). The areas and items to check include:

1. Gases - pressures, carrier gas average linear velocity, and flow rates (detector, split vent, septum purge). 2. Temperatures - column, injector, detector and transfer lines. 3. System parameters - purge activation times, detector attenuation and range, mass ranges, etc. 4. Gas lines and traps - cleanliness, leaks, expiration. 5. Injector consumables - septa, liners, O-rings and ferrules. 6. Sample integrity - concentration, degradation, solvent, storage. 7. Syringes - handling technique, leaks, needle sharpness, cleanliness. 8. Data system - settings and connections.

GHOST PEAKS AND CARRYOVER:

System contamination is responsible for most ghost peaks or carryover problems. If the extra ghost peaks are similar in width to the sample peaks (with similar retention times), the contaminants were most likely introduced into the column at the same time as the sample. The extra compounds may be present in the injector (i.e., contamination) or in the sample itself. Impurities in solvents, vials, caps and syringes are only some of the possible sources. Injecting sample and solvent blanks may help to find possible sources of the contaminants. If the ghost peaks are much broader than the sample peaks, the contaminants were most likely already in the column when the injection was made. These compounds were still in the column when a previous GC run was terminated. They elute during a later run and are often very broad. Sometimes numerous ghost peaks from multiple injections overlap and elute as a hump or blob. This often takes on the appearance of baseline drift or wander. Increasing the final temperature or time in the temperature program is one method to minimize or eliminate a ghost peak problem. Alternatively, a short bake-out after each run or series of runs may remove the highly retained compounds from the column before they cause a problem. Performing a condensation test is a good method to determine whether a contaminated injector is the source of the carryover or ghost peaks.

GC Troubleshooting
Excessive Baseline Noise
Possible Cause Injector contamination Column contamination Detector contamination Solution clean the injector Comments gas lines may need cleaning

Limit bakeout to 2 hrs. Bakeout the column Bonded and crosslinked Solvent rinse the column phases only clean the detector noise increases over time, and not suddenly

Contaminated or low quality gases

use better grade gases check for gas leaks and expired gas traps

usually occurs after changing a gas cylinder consult literature usually at the column fittings or injector use new part may require up to 24 hrs to fully stabilize critical for trace level analyses

Incorrect detector gas flow adjust flow rates to rates recommended values Leak when using MS, ECD, or TCD Old detector filament, lamp, or multiplier Un-equilibrated detector find and eliminate the leak replace appropriate part allow detector to stabilize

Incompletely conditioned fully condition column column

Change in Peak Size

Tailing Peaks
Possible Cause Column contamination Solution Trim the column Solvent rinse the column Irreversible Change sample solvent Install a retention gap Comments Trim half to 1 meter from the front of the column Only for bonded and crosslinked phases Only affects active compounds More tailing for the eluting peaks, or those closest the solvent front 3 to 5 meter retention gap is sufficient

Column activity

Solvent-phase polarity mismatch Solvent effect violation for splitless or on-column injections Too low of a split-ratio Poor column installation Some active compounds always tail

Peak tailing decreases with Decrease the initial retentiongas lines may need column temperature cleaning Increase the splitratior Reinstall the column None Flow from split vent should be 20ml / min or higher More tailing for the early eluting peaks Most common for amines and carboxylic acids

Retention Time Shift

Possible Cause Change in carrier gas velocity Change in column temperature Change in column dimension Large change in compound concentration Leak in the injector Blockage in gas line Solution Check gas velocity Check column temperature Verify column identity Comments All peaks will shift in the same direction by approximately the same amount Not all peaks will shift by the same amount Switched column?

Use a different May also affect adjacent peaks sample concentration Leak-check the injector Clean or replace Achange in the peak size occurs also Most common for split line. Check

clogged line

flow controllers and solenoids

Split Peaks
Possible Cause Injection technique Mixed sample solvent Poor column installation Sample degradation in the injector Solution Change technique Change to single solvent Reinstall column in the injector Reduce the injector temperature Change to an oncolumn injection Comments Erratic plunger depression or having sample in needle Worse for solvents with large differences in polarity or boiling points Usually a large error in the insertion distance Peak broadening or tailing may occur if the temperature is too low Requires an on-column injector

Excessive Baseline Noise

Possible Cause Injector contamination Column contamination Detector contamination Contaminated or low quality gases Solution clean the injector Comments gas lines may need cleaning

Limit bakeout to 2 hrs. Bakeout the column Bonded and crosslinked Solvent rinse the column phases only clean the detector use better grade gases check for gas leaks and expired gas traps noise increases over time, and not suddenly usually occurs after changing a gas cylinder consult literature usually at the column fittings or injector use new part may require up to 24 hrs to fully stabilize critical for trace level

Incorrect detector gas flow adjust flow rates to rates recommended values Leak when using MS, ECD, or TCD Old detector filament, lamp, or multiplier Un-equilibrated detector find and eliminate the leak replace appropriate part allow detector to stabilize

Incompletely conditioned fully condition column

column

analyses

Change in Peak Size

Possible Cause Solution Comments All peaks may not be equally affected.May be caused by system contamination and not the detector Decrease column temperature and check for appearance of a peak shoulder or tail

Check gas flows, Change in detector temperatures, and response settings.Check background level or noise Co-elution Change in the purge activation time Change in the injection volume Change column temperature or stationary phase

Check the purge activation For splitless injection time Check injection technique Injection volumes are not linear Sample degradation, variances in sample temperature or ph Sample leaks passed the plunger or around the needle, Leaks are often not readily visible Remove about half to 1 meter from the front of the column. Only bonded and crosslinked phases Only affects active compounds

Change in sample Check and verify sample concentration concentration Leak in the syringe Use a different syringe

Column contamination Column activity

Trim the column Solvent rinse the column Irreversible

Tailing Peaks
Possible Cause Column contamination Solution Trim the column Solvent rinse the column Irreversible Change sample solvent Install a retention Comments Trim half to 1 meter from the front of the column Only for bonded and crosslinked phases Only affects active compounds More tailing for the eluting peaks, or those closest the solvent front

Column activity Solvent-phase polarity mismatch

gap Solvent effect violation for splitless or on-column injections Too low of a split-ratio Poor column installation Some active compounds always tail

3 to 5 meter retention gap is sufficient

Peak tailing decreases with Decrease the initial retentiongas lines may need column temperature cleaning Increase the splitratior Reinstall the column None Flow from split vent should be 20ml / min or higher More tailing for the early eluting peaks Most common for amines and carboxylic acids

Retention Time Shift

Possible Cause Change in carrier gas velocity Change in column temperature Change in column dimension Large change in compound concentration Leak in the injector Blockage in gas line Solution Check gas velocity Check column temperature Verify column identity Comments All peaks will shift in the same direction by approximately the same amount Not all peaks will shift by the same amount Switched column?

Use a different May also affect adjacent peaks sample concentration Leak-check the injector Clean or replace clogged line Achange in the peak size occurs also Most common for split line. Check flow controllers and solenoids

Split Peaks
Possible Cause Injection technique Mixed sample solvent Poor column Solution Change technique Change to single solvent Reinstall column in Comments Erratic plunger depression or having sample in needle Worse for solvents with large differences in polarity or boiling points Usually a large error in the insertion

installation Sample degradation in the injector

the injector Reduce the injector temperature Change to an oncolumn injection

distance Peak broadening or tailing may occur if the temperature is too low Requires an on-column injector

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