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The CHROMacademy Essential Guide to
GC Troubleshooting - Gas Supply and Inlet Issues.

The Essential Guide from LCGC’s CHROMacademy presents the first in our series of webcasts on Practical GC Troubleshooting. In this session, Dr. John Hinshaw (Senior Scientist , BPL Global Ltd) and Tony Taylor (Technical Director, Crawford Scientific), present practical troubleshooting and maintenance information associated with gas supply and management, autosamplers and split / splitless sample inlets. This session considers good practice for gas supply and delivery, electronic pressure control issues and gas quality management. We also investigate common problems and mistakes in choosing sample solvents and issues with autosamplers and syringes. We consider the most common instrument problems and mistakes associated with split / splitless inlets and examine essential maintenance to help avoid the pitfalls. We include examples of the impact of all issues considered on chromatography and quantitative data analysis and consider various troubleshooting tests and diagnostics that might be used. A must see for everyone working with GC instruments.


 
Presenters: john hinshaw John Hinshaw
Senior Scientist
BPL Global Ltd
tony taylor Tony Taylor
Technical Director
Crawford Scientific

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Topics include

  • Gas supply management and good practice
  • Pressure management issues
  • Sample preparation and autosampler problems
  • Common Split / Splitless inlet problems
  • Chromatographic, instrument and quantitative symptoms
  • Diagnostic tests
  • Common gas supply and inlet maintenance operations
 

Who Should Attend:

  • Anyone who uses GC equipment and who wants to improve their troubleshooting and instrument maintenance skills.

Key Learning Objectives:

  • Recognize the importance of gas management on GC system performance
  • Explore practical aspects of GC sample introduction
  • Understand the importance of keeping your GC system and consumables (gases, liners, septum, syringes) in good working order
  • Provide personnel with a thorough understanding and knowledge of troubleshooting GC leaks
  • Troubleshoot everyday problems commonly found with split/splitless inlets
  • Implement basic troubleshooting and maintenance for GC autosamplers

The Camtasia Studio video content presented here requires JavaScript to be enabled and the latest version of the Adobe Flash Player. If you are using a browser with JavaScript disabled please enable it now. Otherwise, please update your version of the free Adobe Flash Player by downloading here.

 

If you have any more questions on this or any other topic - please post them on our forum >>

The CHROMacademy Essential Guide Tutorial
GC Troubleshooting - Gas Supply and Inlet Issues

This session considers good practice for gas supply and delivery, electronic pressure control issues and gas quality management. We also investigate common problems and mistakes in choosing sample solvents and issues with autosamplers and syringes. We consider the most common instrument problems and mistakes associated with split / splitless inlets and examine essential maintenance to help avoid the pitfalls.

Topics include

  • Gas supply management
  • Pressure management issues
  • Sample preparation and autosampler problems and symtoms
  • Inlet problems, Chromatographic and Instrument Symptoms and Diagnostic Tests
  • Common Preventative Maintenance Operations
  • References - Resources

 

 

  ask the CHROMacademy experts

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Gas Cylinder, Regulator and Tubing - Maintenance and Good Practice

Cylinders

Due to the pressures required and the volume of gas typically consumed, gas cylinders are typically employed to supply GC instruments. [1]
More recently, gas generators have been introduced to supply some detector gases.


Figure 1.  Gas cylinders must be properly secured with a chain, strap, or cable to a stationary building support.
 

 

 

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Cylinders operate under high pressure (thousands of psi) and care should be taken when handling - they should not be dropped and should be secured to a wall using a chain or safety strap. It is advisable to use a gas manifold with a back-up cylinder to avoid any interruption in gas flow. This is particularly important for the carrier gas, as if the supply fails whilst the GC column is heating – irreparable damage may be done. Further – a constant flow of air through gas filters (see later) can quickly saturate the filter materials.

 

Regulators

The purpose of a pressure regulator is to maintain constant gas pressure to the GC.[2 - 6] Two types are available – cylinder regulators and line regulators. Cylinder regulators attach directly to the cylinder valve and regulate the pressure from the cylinder (2500psi) down to a more useable line pressure (around 200 - 400psi). Cylinder regulators have two pressure gauges – an inlet (high pressure) gauge which reads the cylinder pressure, and a delivery (outlet) gauge. The final delivery pressure is adjustable by turning the knob on the front of the regulator.

 

 

 

 

 

 

 

 

 

 

Figure 2.  Two stage cylinder regulator.

 

Care should be used when replacing cylinders and the regulator should always point away from you. Certain regulator types have a left hand thread and are made from brass – these are typically used with flammable gases such as hydrogen and are designed to be anti-spark and have diaphragms which are less susceptible to corrosion.

NEVER – use solvents (especially acetone or methanol) to clean the threads of the regulator or those in the cylinder. These represent a spark risk when connecting the regulator to the cylinder.

Avoid Teflon tape – shards of tape can get blown into the regulator, causing a leak, valve malfunction or erroneous reading. Ensure that you blow any dust or particulates out of the cylinder head connection prior to fitting the regulator.

Oxygen and moisture can migrate through elastomeric components and rubber diaphragms and enter the carrier gas - therefore all regulators should have Teflon coated stainless steel diaphragms to minimise carrier contamination.

 

Pigtails are sometimes installed between the regulator and the gas line. They allow the necessary flexibility when replacing the cylinder and are typically constructed of braided stainless steel with a Teflon core.

 

It is possible for moisture and oxygen to permeate through the Teflon and so purifiers (filters) should always be used down line –as close to the GC as possible.

 

Figure 3.  Pigtails.
 

After installing a fresh gas cylinder, the regulator valve should always be fully opened – to prevent the cylinder from shutting down unpredictably (usually within the first 24 hours), due to internal pressure reduction within the regulator.

Important:

As a general rule a cylinder should be replaced when the main bottle pressure reaches 200-300 psi (or 10% of the original cylinder pressure). As the cylinder pressure drops, impurities such as moisture, hydrocarbons and small particulates concentrate within the gas – drastically reducing the gas purity.

 

Safe laboratory practice should be used to transport and use gas cylinders under high pressure:

  • Never move a cylinder with a regulator installed
  • Ensure safety caps are in place over the gas valve when transporting a cylinder
  • Always chain or strap cylinders to stationary objects or walls while or use OR IN STORAGE
  • Always use cylinder condition labels to show whether it is FULL, IN USE or EMPTY
  • Always leave at least 200psi residual gas in a depleted cylinder.  Store the empty cylinder in a storage area with the valve closed
  • Do not expose cylinder to temperatures exceeding 125oF
  • Work in pairs to change a cylinder
  • Use only approved cylinder trolleys with safety restraints to move cylinders longer distances
 

 

 

 

 

 

 

Figure 4.  Specially designed trolleys must be used when moving
high pressure cylinders from one place to another.

Air compressors can be used to supply air, however most compressed air contains trace hydrocarbons from lubricating oil used in the compressor. Compressed air that contains hydrocarbons or sulphur gasses is not recommended for operating FID, FPD, TCS or ELCD detectors. It is advisable to use filters and purifiers to remover hydrocarbon residues from the compressed air source. Modern air generators are available which produce and filter the air within a single unit.

Nitrogen generators are also available which use membrane or carbon bed technology to filter Nitrogen from a compressed air supply – leaving the resulting gas moisture, oxygen, hydrocarbon and phthalate free.

 
 

 

Figure 5.  Pressure regulator-pig tail connection.
 
 
Figure 6.  Automatic switchover systems ensures a continuous pressure supply.
 

It is essential to use clean chromatographic grade tubing – which has been solvent washed to remove residual hydrocarbons (grease) used in the drawing process. These contaminants can migrate to the gas stream causing noise and elevated baselines with some detectors.

 

 

 

 

 

 

 

 

 

Figure 7.  GC coiled tubing is used to achieve flexibility in installation.

 

The use of Teflon, plastic, PTFE, PVC or Tygon tubing should be avoided at all cost as all are permeable to oxygen and moisture – causing damage to the GC column if these substances diffuse into the gas stream – especially when the column is at elevated temperature. Most of these tubing types will also generate impurities that can cause ghost peaks and baseline instability.

 

Gas Purity

Clean carrier gas is essential to prolong the lifetime of GC columns, and is required to achieve less noisy baselines and good peak shape. Detector gases are also susceptible to impurities which lead to increased background signal, baseline noise and reduced sensitivity.

The three main gas contaminants of concern are Oxygen, Moisture and Hydrocarbons. The first two can enter the gas stream through permeation of tubing and fittings, the latter can arise from grease and lubricating oils within tubing, gas compressors used to generate air or plastic tubing used in gas generators. Both oxygen and moisture degrade the column stationary phase through oxidative degradation and shorten column lifetime. Hydrocarbons cause ghost peaks, increase baseline noise and reduce detector sensitivity.

Gas suppliers all have proprietary names for high quality gas – however in general they all carry products that can be categorized as ‘Certified’ and ‘High Purity’. Wherever possible the gas supplied to the GC should contain less than 1ppm of the relevant impurity and be designated as 99.999% pure or better.

 

Gas Traps / Filters

To reduce gas contamination, high purity gases are used in conjunction with gas purifiers (‘traps’). The traps are fitted as close as possible to the GC instrument to reduce contamination between the trap and the instrument. Table 1 shows which trap types are recommended for the various gas supplies and detectors typically used in GC.

Gas stream Recommended purifier
Carrier gas Hydrocarbon, Moisture, Oxygen
FID / NPD / FPD (Fuel Gas) Hydrocarbon
FID / NPD / FPD (Make-up Gas) Hydrocarbon
FID / NPD / FPD (Oxidiser) Hydrocarbon
ECD (Reaction and Make-up Gas) Moisture and Oxygen
TCD (Make-up) Hydrocarbon

Table 1.  Recommended purifiers for various gas streams.

Hydrocarbon traps help to remove contamination arising from solvent, oil and grease and are generally constructed from activated charcoal.

Older style traps are constructed from steel, which are robust; however suffer in that they are non-indicating. That is they do not contain a substance that changes colour to indicate that the trap is exhausted.

Molecular sieve traps are used to trap water vapour thus increasing column and oxygen trap lifetimes.  Molecular sieves are known to reduce baseline noise from sensitive detectors such as ECDs and mass spectrometers.

 

 

 

 

 

 

Figure 8. Molecular Sieve S-trap.

 
 

Modern Hydrocarbon traps are self-indicating and available in cartridge format.  These traps plug into a base plate and can be conveniently changed without interrupting the gas flow.  Indicating traps are typically constructed from glass or impermeable plastics which do not allow the passage of oxygen and moisture.

 


Figure 9.
 Hydrocarbon trap.  Courtesy of SGE.

 
 
 

 

Figure 10. Impure carrier can cause noisy baselines, high background and poor sensitivity.
 
 

Modern hydrocarbon traps have capacities of around 6-12 grams (measured as n-butane) and will typically reduce the carrier hydrocarbon content to less than 100ppb.

Removal of hydrocarbons from the carrier and detector gases ensures a stable baseline with low noise levels and detection limits.

Moisture traps usually contain molecular sieve that is heat treated and packed under vacuum. Removing moisture from the carrier and detector fuel gases prolongs column lifetime and ensures low noise baselines with good detector sensitivity. The moisture trap will also protect the oxygen filter further down the gas line.

The indicator for modern moisture traps have been altered from the traditional cobalt blue dye according to European Safety directives. Newer traps use a silica based indictor which changes from orange/brown to off-white when exhausted.

   

Figure 11. Moisture trap.  Courtesy of SGE.

   
 

Modern moisture traps have capacities of around 7-10 grams (H2O) and will typically reduce the carrier hydrocarbon content to less than 1ppm.

Oxygen contamination in the carrier gas can produce excessive column bleed at elevated column temperatures caused by oxidative degradation of the stationary phase.

Oxygen traps usually contain aluminium oxide which has a high capacity for getting and binding (fixing) moisture.

Modern oxygen traps have capacities of around 1000 mL (O2) and will typically reduce the carrier hydrocarbon content to less than 50ppb.

 

Figure 12. Oxygen indicating trap.  Courtesy of SGE.

 

Figure 13. Stainless steel oxygen trap.  Courtesy of SGE.

 
 
 

Figure 14. Oxygen contaminated carrier gas can cause severe column bleed at elevated temperature.

 

Figure 15. Carbon dioxide trap (used for specific applications but commercially available).

 
 

Depending on the number (and location) of GC instruments, there are two trap configurations:

  • Install the traps on the main line, thus all GC systems are fed with gas purified by the same traps
  • Install a set of traps at each GC system (each GC system has its own set of traps)

In the first case, it may be cost effective to install a set of large capacity traps in order to minimize the frequency of replacement.

The traps should be installed in accordance to your supplier’s indications (vertical position, required fittings, etc). If multiple traps are used, the order of the traps starting from the gas source should be:

  • Moisture
  • Hydrocarbon
  • Molecular Sieve
  • High capacity oxygen
  • Indicating oxygen
 

Please bear in mind that:

  • One or two of the aforementioned traps may not be needed
  • Sometimes additional traps (such as carbon dioxide or sulphur) should be used
  • Traps should be installed as close to the GC unit as reasonably possible
  • Gas purifiers should be installed in a vertical position

The diagram below illustrates GC traps configuration.

Figure 16. GC traps configuration.  Remember that gas purifiers should be installed in a vertical position.
 
 
 

Figure 17. Combined gas trap unit.  Courtesy of Agilent Technologies.

 
Trap Capacity (g) Efficiency (ppb) Dimensions (cm) Weight (kg)
Oxygen (high capacity) 3.5 < 10 5.0 x 37 1.2
Oxygen indicating 0.05 < 0.1 3.2 x 26 0.2
Moisture 12 < 10 3.2 x 26 0.3
Hydrocarbons 8 < 1 5.0 x 37 1.0
Table 2.  Typical trap capacities.
 

Make sure you know the recommended operating conditions for your trap.  However, in general terms, most GC traps are designed to operate at:

  • room temperature
  • top flow rates not exceeding 1-2 L/min
  • maximum operating pressure below 100-200 psi

Trap capacities depend upon manufacturer, size, etc.  For more information consult your favourite GC trap manufacturer or provider.

 
Figure 18. Siloxane bleed – O2 catalysed back biting reaction.
 
Figure 19. Impact of moisture on column bleed 50oC to 350oC, 20oC/min.  Courtesy of Agilent Technologies.
 
 
 

When using Mass–Flow sensitive detectors such as FID, NPD or FPD, the reduction in column flow rate has the added disadvantage of changing the chromatographic baseline (background reading) – leading to a steadily rising or falling baseline position. This can make integration of the peaks in the chromatogram difficult and irreproducible.

Using electronic pressure control allows the instrument to compensate for changes in gas pressure as the column oven temperature is increased. This will result in a flatter baseline, better peak shape and shorter elution times for more highly retained analytes.

The chromatogram below, was obtained under constant pressure mode conditions; the initial analysis flow rate of 0.92 mL/min (at 50oC) falls to 0.38 mL/min. at the end of the analysis– the carrier gas pressure is 5 psi.

Figure 20. Constant pressure GC separation of acrylic acid impurities using FID detection.

 

 

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The chromatogram below, was obtained under constant flow mode conditions; unlike in the previous example, the carrier gas flow rate was kept constant to a value of 0.92 mL/min. throughout the whole analysis. The pressure being ramped from 5psi to a final value of 11.5 psi at 300oC.
 

 

 

 

 

 

Figure 21. Constant flow GC separation of acrylic acid impurities using FID detection.

 

The previous figures reveal that the chromatography has been improved in several respects when using constant carrier flow in Temperature Programmed GC. There has been an overall gain in sensitivity (the peaks areas have increased), mainly due to a decrease in peak width. The rising baseline has been eliminated due to the constant flow into the mass flow sensitive detector – allowing more reproducible integration and quantitation of peaks 4 and 5. The retention time has decreased by a factor of three.

 

 

 

 

Figure 22. Electronic Pressure (Pneumatic) control unit (EPC).  Courtesy of Agilent Technologies.

 

  • EPC pressure regulating solenoids and read back can fail
  • Leads to an inability to achieve the set point flow (pressure) OR variable carrier / split / septum purge flow (pressure)

 

 
 

Measuring Gas Flow

As temperature and air pressure may vary from place to place, standard reference conditions for testing, reporting and calibration should be used.  Selected standard reference conditions for gases are listed in the table below:

Reference Pressure (kPa)* Temperature (oC)
STP 100.000 0
NTP 100.325 20
SATP 010.000 25
ICAO 100.325 15
Table 3.  Typical trap capacities.

* Approximately 1 atmosphere (1atm ≈ 101325 Pa ≈ 760 torr ≈ 14.7 psi)

 

Many GC systems express their readings on a standard 25oC and 1 atmosphere basis. Therefore, a flow measured with an external device (such as a bubble meter) may not agree with your instrument reading.[2] Given that flow was measured at local rather than standard conditions. Make sure you know and use the same reference conditions than your GC system uses. In case of doubt, consult your GC manufacturer’s handbook.

Example: When using an electronic flow-meter you find a flow rate of 5 mL/min (measured at 740 torr and 18oC), calculate the equivalent flow rate (also in mL/min) that would appear in the dashboard of your GC system (which uses STP conditions).

Considerations:

  • Use absolute temperatures
  • Make sure that units are consistently used (for example, do not use P1 in bar and P2 in psi)
  • At moderate conditions of temperature and pressure, the so called “ideal gas” law holds

 

Lab Conditions

V1 = 5 mL/min
T1 = 18 oC = (18 + 273.15 ) K = 291.15 K


STP Conditions

VSTP = ?
TSTP = 0 oC = (0 + 273.15 ) K = 273.15 K
PSTP = 100.0 kPa

Therefore:

The table below, lists volumetric flow rates equivalences.

Table 4.  Equivalence of volume measured at (760 torr and 25oC) in different temperature and pressure systems.

 
 

Troubleshooting Flow Rate Problems in EPC Controlled Systems

There are many reasons why GC instrument do not reach the required flow rate (pressure) or a constant carrier gas flow rate is not achievable.  These include:

  • Leaks at the column fitting or inlet septum fitting
  • Insufficient gas supply
  • Wrong system configuration (typically low flows with large internal diameter capillary columns)
  • Problems with the proportioning valves of the electronic pressure control system

The section below, lists some useful tests that can be performed to isolate and identify the problem.

 
What ‘Tests’ Might be Carried out to Ensure the Inlet is Working Properly?

Tests to perform when system does not reach the desired set-point pressure or flow:

  • Check there is sufficient gas supply applied to the inlet
  • Check pressure on gas cylinders and any gas supply lines
  • Pressure supply should be at least 10psi greater than the set-point
  • Make sure the inlet is leak free
  • Use electronic leaks detectors (if possible)
  • Use suitable leak detecting solutions
  • Make sure the system is properly configured
  • Is the flow is too low for the column in use
  • Is the column plugged or not properly installed
  • If using a gas saver mode, make sure the flow rate is high enough to maintain the highest head pressure required during the run
  • The EPC pressure sensor may be broken.  Seek expert advise
  • Split/splitless
  • Split ratio is too low
  • Inlet control valve is stuck.  Seek expert advise
 
 

Tests to perform when system exceeds the desired set-point pressure or flow:

  • The EPC pressure sensor may be broken.  Seek expert advise
  • Split/splitless
  • Split ratio is too high
  • Proportional control valve is stuck closed.  Seek expert advise
 
 

Tests to perform when system pressure or flow fluctuates:

  • Make sure the inlet and column connection are leak free
  • Use electronic leaks detectors (if possible)
  • Use suitable leak detecting solutions
  • Check for system (especially the column) for restrictions / blockages
  • Check correct liner being used and is free from debris
  • Check /change split vent trap as appropriate
  • Avoid large room temperature fluctuations
  • Implement temperature control
  • Re-locate GC system
 
 

Tests to perform when measured and instrument displayed flows are not equal:

  • Use additional flow meters to compare with instrument readings
  • Check correct column being used (length, internal diameter,..)
  • Check correct gas was selected in the instrument firmware
  • Check the split line and split trap is free of restrictions
  • Check for leaks at the septum nut and column connection
 
 

General Leak Testing
Step 1.  In general terms, the maximum pressure that most GC systems can handle safety is 80 psi.  As a rule of thumb, the carrier gas pressure regulator, at the gas cylinder, should be set 20 psi higher than the highest required carrier gas head pressure.

Step 2.  Look for leaks in the lines and connections between the gas cylinders and flow controllers.  In order to perform this step, the GC system should turned off; close each of the gas cylinders one at a time and closely monitor the pressure indicator on the cylinder regulator to see if pressure remains constant within a reasonable time frame (a 5 minutes pressure plateau is a good indication this portion of the GC system is leak free).  If pressure drops significantly during the testing period, then use a leak check solution or a leak detector to verify the integrity of the gas supply lines.

Usually 1:1 mixtures of water and alcohol (methanol, ethanol, propanol or isopropyl alcohol) make useful bubble mixtures.

 
 

Figure 23. Leak testing using a water / alcohol solution.

 

 

Warning: do not use soap and water as a leak checking solution.  Soapy solutions may get inside the tubing or fitting and contaminate the GC system with residues which give rise to raised baselines.

 

 
 

 

Figure 24. Electronic leak detector.

 

 

Alternatively, you can use an electronic  leak detector.  These are very practical and can be used even when testing hot spots, although obviously are a little more expensive.

 
 
 



Figure 25.
 Modern electronic leak detector. 
Courtesy of Ellutia (Cambridge, UK).

 

 

Step 3.  Look for leaks between flow controllers and injection port.  You should start by disconnecting the column and inserting a Swagelok nut with a blanked ferrule (or a paperclip in place of the column will work as a blanking device) and blank the split line outlet with a blanking nut.  With the inlet set to split mode - apply a column flow / pressure via the instrument and note the instrument head pressure.  Close the line regulator into the instrument.  The instrument head pressure reading should not drop appreciably (>5%) over a two minute period if there are no leaks within the inlet or inlet connections.

Step 4.  Reinstall the GC column and check for leaks in the same manner described in step 2.

 
 

 

Choosing an Appropriate Sample Solvent

The GC sample solvent, usually a lower-boiling point organic solvent, should be capable of dissolving all of the analytes of interest within the sample.

 

 

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Solvent Density (g/mL) * Mol. Weight (Da) Boiling Point (oC) ** VE (mL/mL) ***
Isooctane 0.69 114 99.3 138
Hexane 0.66 86 68.5 174
Toluene 0.87 92 110.6 196
Pentane 0.62 72 36 198
Ethyl Acetate 0.9 88 77.1 233
Chloroform 1.48 119 61.2 284
Methylene Chloride 1.33 85 39.6 356
Methanol 0.79 32 65 563
Water 1 18 100 1261

Table 5.  Estimated volume expansion (VE) at 250oC and 190 kPa

* Measured at 25oC
** Measured at 1 atm
*** Interpretation: 1 mL of liquid hexane would assume 174 mL when vaporized under 250oC and 190 kPa.

The selection of the GC solvent should be done by considering not only the injection strategy but the detection means used for the analysis.

Split Injection: ensures that the sample is rapidly volatilized and transferred to the capillary column – hence ensuring a narrow analyte band. For this reason initial column temperatures for split injection tend to be higher than the boiling point of the sample solvent.

Splitless Injection:  suited for the analysis of trace levels of higher-boiling solutes that can be injected in a low-boiling solvent, under conditions where the solvent condenses on the column just before the solutes are transported into that region.  Remember that the initial temperature of the column should be at least 10oC below the boiling point of the sample solvent.

GC-MS:  selection of solvents should consider the boiling point of all analytes within the sample.

 
Boiling Point Typical Solvents MS Analysis
Analyte of Interest GC Solvent
High (over 140oC) Low Dichloromethane Detect high-boiling analytes.  Set the MS instrument ON after the low boiling solvent has passed
Low (below 80oC) High Xylenes Detect low-boiling analytes.  Set the MS instrument OFF before the high boiling solvent begin to pass
Table 6.  GC-MS solvent selection.
 
 

Injection Volume

There are many factors that govern the volume of GC sample to be injected:

  • Concentration (amount of analyte per injection)
  • Column type (capillary, packed, megabore)
  • Injection type (split, splitless,…)
  • Liner dimensions
  • Solvent type (expansion volume)
  • Inlet head pressure and temperature

Due to the high temperature conditions within the inlet port, injected samples undergo a rapid volumetric expansion.  If the sample’s expanded volume is larger than the injection port (liner) volume, it flashes back up to the top of the injection port and out of the gas lines both coming into and leaving the inlet- where sample components will condense.  This unwanted situation can create a multitude of problems including:

  • Sample loss
  • Poor resolution
  • Peak shape problems (peak splitting, tailing, etc)
  • Carry-over
  • Ghost peaks

Backflash can be prevented by:

  • keeping the injection port at the lowest reasonable temperature
  • using liners of large volume
  • injecting the minimum sample volume that yields the required detection and quantitation limits
  • using injection solvents of high molecular weight (if possible)

The nature and volume of the sample solvent injected into the split/splitless inlet will have a major effect on the accuracy and reproducibility of quantitative analysis and the chromatographic peak shape. As the injection is made, the sample solvent rapidly volatilises and expands into the gas phase. To avoid quantitative problems, the total volume of the gas should be able to be constrained within the volume of the inlet liner. If this is not the case, then the excess gas will spill over into the inlet gas supply and septum purge lines. The temperature in these lines rapidly decreases, and it is possible for the sample solvent vapour (containing the analyte), to re-condense, ultimately depositing analyte onto the inner walls of the tubing.

When the next ‘overloaded’ injection is made, the sample solvent from this injection will again ‘backflash’ into the gas lines. In this instance analyte deposited during the previous injection will be ‘lapped’ back into the inlet – ultimately finding its way onto the column. This will cause ‘carry-over’ and will reduce quantitative accuracy and reproducibility.  More information regarding ‘backflash’ will be given in the pages to follow.

Column capacity is defined as the maximum amount of sample that can be loaded onto a column without separation efficiency and peak integrity being compromised – usually determined using peak asymmetry measurements.  As a rule of thumb, column capacity will increase with the amount of stationary phase.

Packed columns tolerate larger amounts of analyte than their capillary counterparts – typically up to a few hundreds of micrograms (of analyte) per injection; capillary column loadability will not usually exceed 30μg per component.

The table below reports approximate sample capacity for selected GC columns.[7]  For more accurate data, please consult your column manufacturer

 
Column ID (mm) Film Thickness (μm)
0.1 0.25 0.5 1.0
0.10 10ng 30-40ng 50-70ng 100-200ng
0.18 20-30ng 60-80ng 100-150ng 250-350ng
0.25 30-40ng 125-175ng 175-250ng 400-500ng
0.32 50-70ng 200-250ng 250-350ng 600-800ng
0.45 80-100ng 300-400ng 400-500ng 800-1000ng
0.53 100-120ng 400-500ng 500-700ng 1000=1500ng

Table 7.  Approximate GC sample capacity as a function of column diameter and film thickness (note that amounts are PER ANALYTE not per sample)

* Where 1 ng = 10-9 g

One might use Table 7 to calculate the sample mass injected on a per analyte basis.  If a sample contains an analyte of interest at approximately 1% and a 0.32mm x 0.5mm i.d. column is being used then the maximum sample load would be 250 – 350mg

 
 

Syringes for GC

The injection syringe plays a crucial role delivering an accurate and precise sample volume.

Syringe Volume:  Chose a syringe so that the injected amount is no smaller than 10% of the syringe nominal volume.[8, 9]  That is no less than 0.5μL for a 5mL syringe and no less than 1μL for a 10μL syringe.  The will keep quantitative errors to a minimum.

Needle style (Fixed or removable):  The fixed style of needle is preferred for experienced operators, autosampler injections and for applications requiring very low detection limits.  These syringes contain needle and stainless steel fixing that is cemented onto the bottom of the glass syringe barrel and have the lowest dead volume of all syringe types.  Fixed needle designs also show the lowest degree of carryover as the space between the barrel and needle is filled with cement.

 
 

Figure 26. Fixed needles.

 

Figure 27. Manual 10μL syringe with plunger protection for manual GC with fixed needle and bevel tip.  Courtesy of SGE.

 
 

Removable needles are recommended for use with inexperienced operators or where the risk of needle bending is higher.  The syringe has a screw thread or Luer fixing at the end of the barrel onto which a housing with an interchangeable needle can be attached.  These needles are more economic for use where needle bending can be common but have lower accuracy and precision.

 
 

Figure 28. Removable needles.

 

Figure 29. Manual 10μL syringe with removable needle and site hole tip for Varian 8200 autosampler.  Courtesy of SGE.

 
 
 

Figure 30. Syringe needle.

 

Figure 31.Manual 5mL headpace syringe delivered with probe and needle.  Courtesy of SGE.

 
 
 

 

Figure 32. Autosampler SGE 0.5μL syringe with removable needle for Agilent Technologies autosampler 7673, 7683 and 6850ALS.  Courtesy of SGE.
 
 

Needle Outside (OD) and Inside Diameter (ID): 

The inside diameter should be selected to ensure minimal dead volume without compromising the ability of the syringe to draw samples of normal viscosity. Medium to high viscosity samples should be diluted or a larger ID needle may be required.   To reduce the possibility of bending, choose the widest available needle outside diameter suitable for the application. Autosampler syringes with 0.63mm OD needles, should be selected for all applications except on-column injection.

Gauge Nominal OD (mm) Nominal ID (mm)
26s 0.47 0.11
26/23 0.47/0.63 0.11
26 0.47 0.22
26 0.51 0.25
23s 0.63 0.11
23 0.63 0.22
22s 0.72 0.18
22 0.72 0.38

Table 8.  Typical needle Internal/External diameter combinations.

 

Needle Point Style: 

The cone shaped needle style has been developed for repeated use with an auotsampler device –which will impact the inlet septum in the same place during each injection and can ‘part’ the septum during the injection to reduce coring or splitting of septa.  Bevelled point needles are recommended for manual injection as they will reduce septum damage in the situation where the septum is not pierced in exactly the same spot each time.

The table below lists and illustrates the use of selected needle tips currently used with chromatographic techniques.

Tip Figure Typical Use
Bevel General purpose tip style.  It is the preferred option for manual injection where piercing the septum in exactly the same place is difficult
Cone Well suited for multi-injection
Dual Gauge Suitable for megabore on-column injection
Dome For use with pre-drilled septa
Site Hole Ideal for large volume gas injection
Sheathed/Bevel Similar to the bevel type but it has stronger mechanical integrity

Table 9.  Typical needle tip styles and uses.[10]

In spite of the wide variety of needle styles, the vast majority of GC applications are dominated by the bevel type. 
 
 

Loading the Syringe – Adequate Washing and Flushing of a Syringe

A syringe should be flushed with approximately 5-10 times its total capacity to eliminate carryover between samples.  This is achieved by repeatedly drawing and expelling solvent/sample from the syringe.  To avoid contaminating the sample, the first 2-3 washes should be discarded to waste.

Small air bubbles can be removed by repeatedly drawing sample into the barrel and rapidly expelling the sample while keeping the needle tip immerse in the solution.  Turning the syringe barrel upright while expelling the sample may also help remove bubbles.

Syringe Care and Maintenance

As previously stated, the integrity of the syringe is of overriding importance to GC operation.  Look for cracks, bent/blocked needles and signs of contamination.  Regular maintenance is crucial for ensuring robustness and long life.

The nature of the contaminant will determine the syringe cleaning agents/protocol to be used.  Solvents commonly used for cleaning purposes include acetone, methanol, acetonitrile and methylene chloride.  Surfactant materials are also used to clean the barrel.

 

Important:

Do not immerse the entire syringe in the cleaning solvent as this may compromise its integrity (by dissolving any adhesive used to bond parts of the syringe).  Clean externally with a tissue.

 

Cleaning Steps:

  • Rinse with a suitable solvent (based on the polarity of the analytes and/or matrix)
  • Rinse with distilled water
  • Flush with acetone
  • Remove plunger and wipe with a tissue
  • Refit the plunger and flush with acetone
  • Allow syringe to dry
 

Important:

  • Never forced the plunger
  • Nano-volume syringes should be clean according to the manufacturer’s instructions!
  • Certain syringes can be heated or even autoclaved
High temperatures can adversely affect the integrity of your syringe (consult your syringe manufacturer); however, as a rule of thumb:
  • Fixed needle syringes can be heated to around 70oC
  • Removable needle syringes can be heated to around 120oC
 

Blocked Needles

  • Do not force the plunger when the needle is blocked
  • Remove the plunger and fill the syringe with solvent
  • Insert the plunger and gently pushed solvent through the needle
  • If blockage persists, then use a cleaning wire
  • Automatic heated syringe cleaners will remove organic residues
  • Replace needle/syringe if everything fails
 

 

 

 

 

Figure 33. Syringe cleaner.  (Courtesy of SGE).

Leakages around the needle tend to develop, especially when the needle is fitted incorrectly.  Remove and refit the needle.  Check the seal is in place and undamaged.

 
 
Symptom Cause Potential Remedial Action
Additional peaks Sample preparation Improve sample clean up
  • Pressure surge
  • Band broadening
  • Irreproducible peaks
Blocked tube
  1. Isolate blocked line, then clean or replace
  2. Install inline filter before valve
  3. Filter sample
  • Bent needles
  • Missed injections
Wrong vial Use correct vials
Additional peaks Contaminated vials / syringe
  1. Clean vials
  2. Use only glass vials
  3. Clean syringe according schedule given above and ensure a suitable autosampler syringe wash is used
Additional peaks Contaminated septa
  1. Use correct septa
  2. Install laminated septa, Teflon side down
Variable peaks intensity Leaky septa
  1. Check for proper seal
  2. Use laminated septa
Broken vials
  • Tray misalignment
  • Wrong vials
  1. Align tray
  2. Use proper vials
  • Wrong sample injected
  • Bent needles
  • Broken vials
Tray misalignment
  1. Align tray
  2. Use proper vials
  • Peak with small intensity
  • No peaks
Blocked needle
  1. Use a cleaning wire
  2. Clean syringe
  3. Implement sample filtration
  4. Replace needle
Bent needle Bent needle
  1. Align autosampler with inlet
  2. Replace needle
Constant error in results Calibration problems
  1. Check/adjust sample size or syringe
  2. Check/replace sample loop
  3. Use internal standard
  4. Check for leaks
  • Small sample size
  • Decreased precision
Air leak, air injected
  1. Tighten/replace fittings
  2. Replace cracked syringe
Accuracy and reproducibility problems Volatile sample (boiling sample)
  1. Implement temperature control
  2. Bear in mind that solvents such as diethyl ether or methylene chloride have very low boiling points (34 and 40oC respectively)

Table 10. Troubleshooting the autosampler.[11, 12]

 
 

 

Setting the Split Ratio

The ‘Split Ratio’ describes the ratio of gas flows between the capillary column and the split flow line – and effectively gives a measure of the volume fraction of the sample vapour that will enter the column.

The calculation of Split Flow is shown below. Of course the magnitude of the split ratio will depend on the concentration of the sample injected and the capacity of the capillary column used.

Typical split ratios lie in the range 1:20 to 1:400 meaning that only 1/20 to 1/400 of the sample is injected onto the analytical column.

 

 

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The split ratio is usually adjusted empirically to obtain a good balance between analytical sensitivity and peak shape. If the split ratio is too low peak shape will be broad and may show the fronting behaviour associated with overloading. Of course if the split ratio is too high, too little sample will reach the column and the sensitivity of the analysis will decrease as peak areas decrease.

When using thick stationary phase film columns (>0.5 mm) or wide bore (0.533 mm i.d.) columns, the sample capacity increases and lower split ratios of 1:5 to 1:20 are typical. With very narrow GC columns (<100 mm i.d.) split ratios can be as high as 1:1000 or more.

In most cases the split ratio should give an approximately linear relationship with analyte peak area – i.e. halving the split should halve the resultant peak area – however this is not recommended for calibrating the instrument response! Below a split ratio of around 1:15 reducing the split ratio may not give a linear relationship.

 
 

 

 

 

Figure 34a. Setting the split ratio.

 

Problems with the Split Line
Most GC’s are equipped with a filter (usually deactivated charcoal) in the split line to remove potentially harmful species prior to venting to atmosphere from the split vent.  These filter become blocked over time and can, along with blockages in the tubing itself, cause incorrect (low or fluctuating) or no split flow during the injection.  The tubing union on the split line exit is particularly susceptible as the exit line is effectively both a restriction and a cold-spot for high boiling materials to condense.

Symptoms typically include poor quantitative reproducibility and an overloaded solvent peak with overloaded (fronting / broad) analyte peaks.

 
 

Split/Splitless Injection Volume

The nature and volume of the sample solvent injected into the split/splitless inlet will have a major effect on the accuracy and reproducibility of quantitative analysis and the chromatographic peak shape. As the injection is made, the sample solvent rapidly volatilises and expands into the gas phase. To avoid quantitative problems, the total volume of the gas should be able to be constrained within the volume of the inlet liner. If this is not the case, then the excess gas will spill over into the inlet gas supply and septum purge lines. The temperature in these lines rapidly decreases, and it is possible for the sample solvent vapour (containing the analyte), to recondense, ultimately depositing analyte onto the inner walls of the tubing.

When the next ‘overloaded’ injection is made, the sample solvent from this injection will again ‘backflash’ into the gas lines. In this instance analyte deposited during the previous injection will be ‘lapped’ back into the inlet – ultimately finding its way onto the column. This will cause ‘carry-over’ and will reduce quantitative accuracy and reproducibility.

 
 

 

 

 

Animation 1. Backflash.
 
 

The expansion volume of the sample solvent is governed by the inlet pressure and temperature, as well as the natural expansion coefficient of the solvent. It is possible to predict the expansion volume and hence the volume of solvent that may be safely injected into an inlet liner of known volume, under set temperature and pressure conditions.

A technique known as ‘pressure pulsed’ injection may be used, in which the inlet pressure is raised during the sample injection cycle. This constrains the expansion of the solvent within the inlet liner and allows for larger injection volumes.

Figure 34 is a useful calculator which allows you to assess the possibility of backflash with your current hardware and operating conditions.

Figure 34b. Optimizing injection volume using a simple backlash calculator

 
 

Inlet Considerations

During splitless injection, it is of vital importance that the inlet is purged of residual vapours once the analyte has been transferred to the capillary column. If this is not done, the solvent peak will show a high degree of tailing and the GC baseline signal may be noisy and rise markedly as the analysis progresses. This is due to the slow bleed of excess solvent and sample (not analyte) components from the inlet into the capillary column. An example showing a splitless injection in which the inlet is not purged is shown below.

 
 

 

Animation 2. Split/splitless injection (inlet is not purged).
 

The inlet purge is achieved by actuating the split (purge) valve that allows a high split flow through the liner, which quickly purges the residual solvent vapours from the inlet. The split flow is high as the aim is to quickly purge the inlet – split flows of 100-200 mL/min. are typical. The time from the beginning of the injection to the time at which the split line is turned on is known as the splitless or purge time.

 
 

What Happens if the ‘Split on’ or ‘Purge’ Time is Too Short?

It is vital that the splitless time is optimised for each application. Too short a splitless time will mean that analyte still resident in the liner will be discarded via the split line. This may lead to poor analytical sensitivity and reproducibility.

 
 

 

 

 

 

Figure 35. Splitless time too SHORT –loss of higher boiling analytes.

 

 

 

What Happens if the ‘Split on’ or ‘Purge’ Time is Too Long?

Too long a splitless time will lead to badly tailing solvent peaks, extraneous peaks and a rising baseline – making reproducible integration difficult.

 
 

 

 

 

 

Figure 36. Splitless time too LONG –broad solvent peak and rising baseline.

 

 

 

 

The splitless time is usually empirically optimised by monitoring the peak area of a mid-eluting peak in the chromatogram. The peak area is plotted against the splitless time and a plot of the form shown below should be produced.

 
 

 

 

 

 

Figure 37. Determining optimum splitless time.

 

 

 

 

 
For reproducible analysis the splitless time should be chosen just onto the plateau of the area response curve as indicated. Typical splitless times lie in the region – 20 – 90 seconds.
 
 

Analyte Focussing

The analyte is slowly introduced from the inlet during the whole of the splitless time (the inlet volume may be exchanged as few as two times during this whole splitless period).

This slow sample vapour transfer would result in the analyte band entering the column over a period of 30 – 60 seconds. or so depending upon the exact analytical conditions. This would entirely negate any efficiency gained through the use of capillary columns and the resulting chromatographic peaks would be unacceptably broad.

 
 

 

Figure 38. Normal versus broad GC peaks
 
 

 

 

Animation 3. Sample focusing.

 
 

To overcome these problems Focussing techniques are used, which usually involves setting the initial oven temperature at a suitably low value ensuring that condensation and reconcentration takes place in the column.

Two discrete focusing (“cold trapping” and “solvent effect”) mechanisms can be identified.

 
 

 

 

 

 

Figure 39. Injection of a solution of n-alkanes in methanol on a dimethylsiloxane (non-polar) stationary phase.

 

 

 

 

 
 

Cold trapping – higher boiling analytes are condensed in a tight band in the temperature gradient between the inlet (~250 oC)
and the column oven (~40 oC)

 
 

 

 

Animation 4. Cold trapping.

 
 

Solvent effect – low boiling (more highly volatile) components remain dissolved in the solvent, which also condenses on the inner wall of the GC column at low initial oven temperatures. The solvent slowly evaporates to give a thin concentrated band of analyte.

 
 

 

Animation 5. Solvent effect.

 
 
 

 

 

Figure 40. Injection of n-alkanes in CS2 (boiling point -46oC).  Efficiency increase of the early eluting (more volatile) analytes can be clearly seen –due to solvent effect.

 

 

 

 

 
 

Inlet Discrimination – Quantitative Analysis Issues

The phenomenon of sample discrimination leads to a non-representative sample entering the analytical column compared to the original sample.

Sample discrimination is described in the diagram below, which shows the detector response to an injection of n-alkanes at equal concentration.

 
 

 

 

 

 

 

Figure 41. Discrimination due to differences in boiling point:  Hot split injection of solution containing equal amounts of normal alkanes in hexane.

 

 

 

 

 

The normalised line shows the original sample composition, and the expected response for each of the n-alkanes. The more highly volatile n-alkanes show total recovery, however for C25, only half of the analyte present in the sample is introduced into the column, and the recovery of C37 is less than 25%.

For higher boiling (less volatile) analytes, the residence time of the syringe needle is too short. The analyte will condense on the cold inner and outer surfaces of the needle – prior to it being withdrawn from the inlet. Some less volatile analytes may never properly volatilise and the sample passes the split point (head of the capillary column) as a mixture of sample vapour and non-uniform liquid droplets. Several approaches to the problem have been postulated including:

  • Optimisation of liner geometry and packing materials to promote sample mixing and volatilisation
  • Optimising the injection routine (filled needle, hot needle, solvent flush, air flush, sandwich method etc.)
  • Improved instrument design to reduce fluctuations in split flow

 

In general, the least amount of discrimination is obtained if the injection is performed as rapidly as possible. For this reason, fast autosamplers generally give less discrimination than manual injection.

 
 

 

 

 

 

Figure 42. Discrimination due to differences in boiling point:  Sample containing equal concentrations of n-alkanes (n-C8 to n-C40). Oven: 35oC for 4 min, 35-320oC at 10°/min, 320oC for 5 min.  Carrier Gas: Helium at 5.O mL/min.

 

 

 

 
 
 

 

Animation 6. Sample discrimination.

 
 

Inlet Temperature

Selection of the correct inlet temperature for Split or Splitless inlet operation is vital. It is necessary to have a high enough temperature to ensure efficient and complete volatilisation of all sample components – this will ensure that temperature (inlet) discrimination of the higher boiling (less volatile) components is minimised.

There is an upper inlet temperature limit for each application to avoid thermal degradation (decomposition) of analytes and sample components, which will lead to poor quantitative reproducibility and/or fouling of the inlet liner.

The accepted arbitrary inlet temperature to begin method development for new analytes and applications is 250oC – and this temperature can be used as a general guide in all cases except where a higher or lower temperature is known to be required (i.e. where particularly thermally labile analytes are involved or where the sample is particularly high boiling).

 
 

 

 

 

 

 

Figure 43. Method development –scouting temperature gradient.

 

 

 

With the inlet set to 250oC a ‘scouting’ temperature gradient may be employed which will elute analytes and sample components across a wide range of boiling point values. In this way, the elution temperature of the highest boiling component of the sample may be estimated. The inlet temperature should then be set to at least 50oC above this temperature to ensure efficient sample volatilisation.

There are a number of chromatographic symptoms that may indicate problems with analyte thermal degradation – mostly involving a plateau at the front of rear of the chromatographic peak. If this symptom is seen the inlet temperature may be reduced in 20oC steps until the problem is resolved.

 
 

 

Figure 44. Typical peak shape of analyte Inlet thermal degradation.
 

Optimisation of inlet temperature can be carried out by decreasing the inlet temperature in 20oC steps – once peak shape problems are overcome the reproducibility of the peak area should be confirmed with at least 6 injections giving a satisfactory relative standard deviation (RSD)(<1%).  Both of these checks are required to indicate thermal stability (it can never be truly ruled out!).

 
 

Septa for Split / Splitless Injection

The septum isolates the inlet from atmospheric pressure – allowing the inlet to be pressurised.  The septum is pierced with the injection syringe needle to allow the sample to be injected.

The septum is ‘plastic’ in nature (i.e. it is deformable), and is held under mechanical pressure with a retaining nut - allowing it to seal around the injection syringe needle and maintain inlet pressure during the injection phase.

Care is required with the torque applied to the septum nut. Over tightening the nut will compress that septum and may promote splitting. Under tightening the nut may cause leaks and pressure failures. Many instrument manufacturers recommend having the septum nut ‘fingertight’.

There are many types of septa available, which vary in characteristic – some of these are highlighted opposite. Care should be taken to use the correct septum and syringe combination for optimum performance.

 
 

 

 

 

Figure 45. Septa for split / splitless injection.
 
 
 

 

 

 

Figure 46. Septum selection.
 
 

Septum Problems

The materials used to plasticise the septum bleed continuously (phthalates etc.). In capillary GC the bleed products may give rise to discrete noise peaks and may also result in a rising baseline as shown below. The septum purge flow of the inlet helps to reduce these effects, however correct septum choice with regards to inlet temperature is important.

 
 

 

 

Figure 47. Splitless injection septum bleed profile.

 

 

 
 

 

Figure 48. Split injection septum bleed profile.

 

 

Septa eventually wear through continuous piercing, and will ‘core’ to deposit fragments into the liner.

 
Figure 49. Septa integrity versus injection number (N).
 
 

Eventually the liner will split or core so badly that a pressure seal is no longer maintained during the injection phase. Peak shape will suffer when this occurs and the baseline shifts are diagnostic of a worn septum.

The figure below shows a typical chromatographic symptom created by a leak at the septum during injection. The baseline position changes after the elution of a large (usually solvent), peak. This indicates that the septum is not sealing correctly around the needle will therefore leak during injection and for a short time afterwards. This symptom is exacerbated if the diameter of the needle used is too large.

A very badly cored or split septum will leak and fail to maintain system (head) pressure. Without a proper maintenance regime, this may be the first time you realise that the septum is causing the problems!

 

Figure 50. Baseline shifts encountered with leaking septum.

 

Following a basic preventative schedule can prevent most septum related problems

  1. Change septa regularly (users will become familiar with expected lifetimes of septa for particular applications), but daily and with column change for instruments that are used constantly.
  2. Use the correct injection syringe, selecting the correct diameter needle for your particular autosampler and use a cone tip rather than a pointed needle to prevent excessive coring.
  3. Condition septa.  Septa may be conditioned by holding at 50oC above the expected operating temperature for an hour or so.  Cycle through the oven temperature program to be used at least twice after conditioning to clear any residues from the system.
  4. Ensure that the septum nut is tight.  Studies have shown, that septa in an HP5890 GC gave up to 400 more injections at 12 in/lbs torque on the septum nut than at 4 in/lbs.  Syringe needles on modern autosampler systems can easily pierce septa under 20 or more in/lbs of torque before bending.
  5. Ensure that the injection liner is clean.  Shards of septa that fall into the injection liner will constantly produce volatile species, giving rise to baseline instability and increased background during the whole analysis cycle.  Inspect the liner (and clean if necessary), every time you change the septum.
 
 

 

 

Figure 51. Baseline shifts encountered with leaking septum.
 
 

Septum Purge Flow

The materials used to plasticise the septum bleed continuously (phthalates etc.).

 
 

 

 

 

Animation 7. Septum bleed contamination.
 
 

In capillary GC the bleed products may give rise to discrete noise peaks and may also result in a rising baseline as shown below.

 
 

 

 

 

 

 

 

 

 

 

Figure 52. Septum bleed profiles.

 
 

The septum purge flow of the inlet helps to reduce these effects, however correct septum choice with regards to inlet temperature is important.

Following a basic preventative schedule can prevent most septum related problems:

  • Change septa regularly
  • Ensure that the GC 'Septum Purge' is at the correct flow rate
  • Use the correct injection syringe
  • Ensure that the septum nut is correctly tight
  • Ensure that the injection liner is clean
 
 

Liners

The selection and correct use of liners (a cylindrical sleeve placed inside the inlet) is of critical importance in split and splitless GC injection. The liner has many functions which include:

  • To constrain the volatilised components of the sample
  • To allow the sample to be split through excess sample and carrier escaping from the liner outlet (some instrument designs)
  • To cause mixing of the sample vapours with the carrier (split injection)
  • To prevent involatile material from fouling the GC column
  • To avoid analyte thermal degradation
  • To decrease the potential for inlet discrimination

 

All of the above are achieved through a number of features of the liner design. These features are often poorly understood and Figure 53 outlines the main features of typical GC inlet liners for a better understanding, The main variables in GC liners selection are:

  • Liner internal diameter
  • Packed / Unpacked
  • Packing Position
  • Liner internal geometry and features including:
  • Upper Gooseneck
  • Lower Gooseneck
  • Inverted (Jennings) Cup
  • Baffled
  • Deactivation Type
 
 

 

 

Figure 53. GC liners.
 
 

Inlet Liners for Split Injection

Split injection is a fast and efficient way to infuse sample on the GC column.  This injection mode is well suited for both highly concentrated and/or dirty samples.[13 - 15]

Due to the short inlet residence time, split liners are designed in such a way that sample vaporization is maximised.  In other words, split injection liners require large surface areas for sample evaporation, this is usually achieved using packing material, increased internal surface area chambers, tortuous flow paths etc.

Straight tube with glass wool.  GC liner for general use.

 

Cup design.  GC liner for high and low molecular weight compounds.

 

Cup with fused silica wool design.  GC liner for high and low molecular weight compounds.

 

Laminar cup design.  GC liner for high molecular weight compounds.

 

Cyclosplitter.  Dirty samples, many injections before cleaning required.

 

The baffle design induces flow turbulence. 
Reproducible performance but prone to discrimination.  Used for compounds with very with close boiling points.

 

Frit splitter design.  Dirty samples, non-active compounds.

Figure 54. A selection of liner designs for Split injection.

 
 

Inlet Liners for Splitless Injection

Useful for low concentration samples, this technique involves an initial hold time.  Therefore, in general, liners for splitless injection do not require high surface area for sample evaporation.

Straight tube liner.

 

The gooseneck design effectively eliminates any dead volume in the injector and provides excellent injection profile for wide-bore columns. In reversed position it can also be used for on-column injections on 0.53mm columns.

Gooseneck design. Useful in trace analysis.

 

Double gooseneck design.  Useful in trace analysis with active compounds are helps prevent backflash.

 

Cyclo double gooseneck design.  Useful in trace, active, dirty samples.

 

Recessed gooseneck design.  Useful with dirty samples.

Figure 55. A selection of liner designs for Splitless injection.

 
 

The practical implications of selecting the wrong liner are many fold.  Not only inaccurate quantification but sample flashback, peak tailing, irreversible adsorbption and mass discrimination issues are not uncommon with poor liner choice.  A good liner will:

  • minimise mass discrimination by ensuring complete sample vaporization
  • have a volume larger than the total volume of vaporized sample and solvent
  • not react with the sample (deactivated)

 

The addition of quartz wool will increase the vaporization surface area while promoting mixing of sample and carrier gas; on top of that, the use of quartz wool will reduce the incidence of particulate matter entering into the column, thus acting as a crude filter. Besides these benefits glass or quartz wool also has its disadvantages. The wool can become adsorptive especially if some fibers are broken or when it has become dirty. It should be exchanged at a regular basis to prevent chromatographic problems. Avoid the use of glass wool when it is not advantageous.

By using liners packed with a selective adsorption material, such as Tenax ®, Carbotrap ® or Chromosorb ®, the range of components that can be trapped in the liner can be significantly extended towards the more volatile components. With liners packed with these materials, even relatively volatile species (e.g. n-C4 ) can be trapped quantitatively at liner temperatures around or slightly below room temperature. With the addition of sub-ambient cooling, components down to n-C2 can be trapped.

 

Figure 56. Large volume injection of a 30 ppm solution of normal alkanes.  Liner packed with a. Tenax and b. glass wool.

 
 

Liner Volume

The sample gas volume which at one time resides in the injector should in all cases be smaller than the internal volume of the liner. If not, the liner is overloaded and back-flash of the sample into various (metal) parts of the injector may occur. This phenomenon will cause poor injection profiles, bad peak shapes, adsorption and possible sample carry over. Many liners used for direct and split injection have dimensions of about 80mm x 4mm resulting in an internal volume of ca. 1ml. The effective volume available for the sample is however significantly lower as part of the liner volume will always be filled with carrier gas.
 
 

Figure 57. GC-FlD chromatograms of a mixture of n-alkane compounds in hexane using three liners with varying internal diameters.[16]

 

 
 

Liner Cleaning and Deactivation
Inlet liners should be disposed whenever contaminated.  However, sometimes acceptable levels of performance can be achieved by cleaning and deactivating the contaminated liner.

Merely dirty undamaged liners, having neither scratches nor cracks, can be returned to a usable condition just by cleaning them.  Dirty liners may be cleaned by;

  • rinsing with solvents (acetone, methylene chloride,…)
  • exposure to streams of pure gases to remove particulate matter
  • exposure to strong acids and detergents
  • brushing or sonication in order to remove particulate matter

Important things to consider when dealing with GC liners:

  • Wear clean gloves at any time
  • Do not scratch the liner, as the liner may be re-activated
  • Gases coming from compressor units are prone to render oil contamination
  • Use clean streams of gas to dry the liner once the rinsing process was finished
  • Strong mineral acids and detergents can be used in case of extremely dirty liners

Unfortunately, over time, GC liners lose their deactivation becoming less and less suited to deal with active solutes.  A simple cleaning will not return them to a usable condition and a deactivation process is required.

Liners will develop active sites through use.  Analyte molecules will absorbe on such sites leading to peaks with reduced intensity and many other issues including:

  • Irreproducible quantitation
  • Reduced sensitivity
  • Poor peak shape (tailing)
  • Discrimination between polar and non-polar compounds

Therefore, the development of active sites renders the liner unfit to perform its job and deactivation (if possible) or replacement become mandatory.

 
 

How Might One ‘Clean’ an Liner?

Belonging to the group of GC consumables, inlet liners should be disposed whenever contaminated.  However, acceptable levels of performance can be achieved by cleaning and deactivating the contaminated liner.

Merely dirty undamaged liners, having neither scratches nor cracks, can be returned to a usable condition just by cleaning them.  In fact, dirty undamaged liners can be:

  • rinsed with solvents (acetone, methylene chloride,…)
  • exposed to streams of pure gases to remove particulate matter
  • exposed to the action of strong acids and detergents
  • brushed or sonicated in order to remove particulate matter

Important things to consider when dealing with GC liners:

  • Wear clean gloves at any time
  • Do not scratch the liner, as the liner may be re-activated
  • Gases coming from compressor units are prone to render oil contamination
  • Use clean streams of gas to dry the liner once the rinsing process was finished
  • Strong mineral acids and detergents can be used in case of extremely dirty liners

Unfortunately, over time, GC liners lose their deactivation becoming less and less suited to deal with active solutes.  A simple cleaning will not return them to a usable condition.  Here a deactivation process is required.

 

Figure 58. Chromatograms obtained with liners in good and bad working conditions.

 
 

Different means can be used to deactivate GC liners:

  • Thermal treatment (usually above 400oC)
  • Deactivation agents

How Might One ‘Deactivate’ an Liner?

Active sites, on the glass surface of the GC liner, can be eliminated by the use of deactivation agents.  When used properly, proprietary deactivation agents will provide thorough / stable deactivation and increased lifetime of the GC liner.  Please carefully follow the instructions given for your selected reagent.

The following is a generic deactivation protocol that uses silylating reagents.[17]

  1. Place the liner in a screw cap test tube fitted with a Teflon-lined cap
  2. Soak the liner between 8 and 24 hours in HCL 1.0N (do not exceed 24 hours)
  3. Visual inspection of liner and cleaning solution, go to step 2 if required
  4. Remove liner from acid solution and rinse with deionised water
  5. Rinse liner with methanol
  6. Dry liner at 100-150oC
  7. Place the liner in a screw cap test tube fitted with a Teflon-lined cap
  8. Soak the liner around 8 hours with 10% solution of trimethylchlorosilane or dimethylchlorosilane in dry toluene or hexane
  9. Remove liner from the silylating solution and rinse with toluene or hexane
  10. Rinse liner with methanol
  11. Dry the liner at 75-100oC for 30 – 60 minutes
  12. Liner is ready for use

Finally and once again, liner deactivation should be performed according to your supplier’s instructions, in case of doubt, ask!

 
 

Why do Some Liners Contain a Packing Material?  What Can go Wrong With Liner Packing and How can Issues be Avoided?

There are many advantages for using liners with packing materials:

  • Increased the vaporization surface area
  • Adequate mixing of sample and carrier gas
  • Reduced incidence of particulate matter entering into the column

 

The most widely used packing materials for GC liners are glass and quartz.

Wool is often used to increase the injectors heat transfer capacity. The evaporation of higher boiling compounds also requires more energy and in those cases quartz wool is often used. It also collects non-volatile heavy molecular weight residues which might harm the column. Besides these benefits glass or quartz wool also has its disadvantages. The wool can become adsorptive especially if some fibers are broken or when it has become dirty. It should be exchanged at a regular basis to prevent chromatographic problems. Avoid the use of glass wool when it is not advantageous.

 

How Might One ‘Clean’ an Inlet?

The GC inlet port will become dirty and active with its use.  If no remedial actions are taken, this unwanted condition will get worse with time.  Remedial actions include not only simple actions such as replacing consumables (liners, septum, etc) but even complex cleaning/deactivation protocols.

There are different GC inlet cleaning protocols, in general terms, all of them are similar in many ways but differences can arise depending upon manufacturers and models.  A generic split/splitless CG inlet cleaning protocol is provided below.  Please bear in mind the following pointers:

  • This protocol is not bullet proof and sometimes it will not restore the GC inlet to a usable condition
  • This protocol is generic and to be in the safe side, you should consult with your GC system manufacturer
  • This protocol should be used when replacing consumables (liners, septum,…) will not restore your inlet to a usable condition
  • Always wear clean globes and use the appropriate tools (for example, do not use a knife instead of screwdriver)
  • Before starting your cleaning, make sure you are familiar with your GC system and that you know what you are doing.  In case of doubt look for expert advise
  • Make sure you have all required tools, spare parts, consumables, solvents and reactants to do the job
 

Figure 59. Chromatograms obtained with inlets in a) poor and b) good working conditions.[18]

 
 

Generic Split/Splitless CG Inlet Cleaning Protocol

  1. Cool the inlet and when the temperature is low enough (around 70oC), turn the inlet flow off
  2. Remove autosampler (if any)
  3. Remove the weldment assembly that covers the GC liner
  4. Remove the GC liner
  5. Turn off the GC oven and remove the column (place a septa over the injection port end of the column, to avoid oxygen entering into the column)
  6. Remove the any fitting from the bottom of the inlet
  7. The injection port should now consist of just a long metallic tube (make sure that all flow lines and liner have been already removed)
  8. Using a brush and methylene chloride, clean the inlet (move the brush up and down only). Rinse with fresh methylene chloride
  9. Repeat step 8 as many times as necessary, usually 2 to 3 are sufficient to achieve the required levels of cleanliness
  10. Repeat step 8 but use acetone instead of methylene chloride
  11. Repeat step 8 but use methanol instead of methylene chloride
  12. Remove any residual solvent, dab the top of the inlet with a napkin
  13. Make sure the inlet is free of particles, in case of doubt go to step 8
  14. Heat the inlet (65oC) to vaporize any remnants of solvent away
  15. Reassemble the system, use new instrument consumables (ferrule, liner, septum washers, etc) whenever required.  Remember that liners could be cleaned rather than replaced.  When reconnecting the column, severe the small portion where the septa was placed (use a column cutter)
  16. Let the inlet at the temperature set in step 14 (65oC) for 10 to 15 minutes to remove any oxygen in the inlet
  17. Run a blank (for more security run at least two of them) to make sure the system was properly cleaned
 
 

What Happens When the Split Line (or Split Line Filter) Becomes Blocked?

Most problems related with column, split and septum purge gas flow rates will result in changes in peak sizes.  If the split flow is extremely low, then tailing peaks or solvent fronts may occur.  The table below lists lowest split ratios typically encounter with capillary GC.

 
Column Diameter (mm) Lowest Split Ratio
0.10 1:100 - 1:150
0.18 - 0.20 1:20 - 1:25
0.25 1:15 - 1:20
0.32 1:10 - 1:12
0.53 1:3 - 1:5

Table 11. Lowest split ratios typically used with capillary GC.

 
 

 

Preventative Maintenance Schedule for Cylinders and Regulators
Cylinders and regulators should undergo periodic inspection whenever a cylinder is being changed in order to detect damage, cracks, corrosion, pressure issues, etc.  Consult your cylinder, regulator or gas supplier for more information.

Keep a record book with maintenance history of your GC and gas supply system.

 

 

 

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Preventative Maintenance Schedule for the Lines Between the Cylinder and the GC
Visual inspection of GC lines should be performed before each use (ideally on a daily basis), look for signals of corrosion.  Leak /pressures tests should be performed whenever a leak is suspected and on an annual basis.

GC Traps
As a rule of thumb, non-indicating traps should be replaced once or twice a year (according to use).  Indicating traps should be replaced whenever the indicating colour changes or according to manufacturers specification.  Visual inspection of traps should occur daily.

Preventative Maintenance Schedule for Autosamplers / Syringes
Syringes should be check whenever used.  Clean or replace whenever dirt, cracks and fractures are visible.  Replace the needle if clogged or if septa wear is abnormal.

 
Element Procedure
Tubing and connections Check for leaks often.  Replace on a monthly basis
Rotor seal Check integrity on a monthly basis.  Replace every 20,000 injections
Inlet filter Check and replace twice a year

Table 12.  Autosampler maintenance schedule general considerations.

 

Preventative Maintenance Schedule for Septa
Check often and replace accordingly.  Look for visual signs of deterioration and ‘coring’ as well as poor chromatography and low column pressure issues.

 

Preventative Maintenance Schedule for Liners
Check on a daily basis.  Clean or replace whenever dirt, cracks and fractures are visible in the liner or if chromatography has been degraded.

 

Preventative Maintenance Schedule for Inlet Seals and O-Rings

O-rings should be replaced often (on monthly basis).  Replace with the GC liner or when signs of wear or ‘baking’ occur (i.e. the o-ring becomes brittle).
 
 

 

  1. “Gas Supply and Pressure Control” from “Theory and Instrumentation of GC”. CHROMacademy
  2. Agilent 6890 Series Gas Chromatograph. Agilent Technologies, Inc.
  3. Restek Guide to GC Setup. Copyright © 1994, 1998 Restek Corporation.
  4. Agilent Inlet EPC Leak Test. Gas Chromatographs. DE 19808-1610 USA. May 2002
  5. Gas Cylinder Safety Guidelines. Iowa State University Environmental Health & Safety Ames Laboratory Environment, Safety, Health & Assurance Copyright © August 1997
  6. HP 5890 SERIES II. Gas Chromatograph. Service Manual. Edition 1, March 1991. USA.
  7. Alltech Capillary Instruction Manual. M171c, Nov. 2007
  8. “Sampling Techniques” from “Theory and Instrumentation of GC”. CHROMacademy
  9. “Sample Introduction” from “Theory and Instrumentation of GC”. CHROMacademy
  10. “Syringe Selection” SGE Analytical Science. http://www.nxtbook.com/nxtbooks/prodigyplus/sge_syringeselection/#/6
  11. Maintaining your Agilent GC and GC/MS Systems. Agilent Technologies 5989-7612EN. April 2008.
  12. John V. Hinshaw. “Autosamplers —Symptoms and Solutions” GC Connections. LCGC Volume 18 Number 12. Pp 1234-1241. December 2000
  13. “Sample Introduction” from “Theory and Instrumentation of GC”. CHROMacademy
  14. http://www.home.agilent.com/
  15. http://www.restek.com/
  16. Hans G.J. Mol, Hans-Gerd Janssen, Carel A. Cramers, and Udo A.Th. Brinkman. “Large Volume Sample Introduction Using Temperature Programmable Injectors: Implications of Liner Diameter” Journal of High Resolution Chromatography. PP 19-27. Vol. 18. January 1995
  17. http://www.chromspec.com/
  18. CAP Kits for Agilent Technologies GCs. (Column and Accessories Performance Kits). Sigma-Aldrich. USA 2002. http://www.sigmaaldrich.com/analytical-chromatography.html
 
Further reading and resources: *** CHROMacademy Registered users only ***
 
Articles

Using Computerized Pneumatics Part I

Using Computerized Pneumatics Part II

A Split Decision

When Peaks Collide Part I

When Peaks Collide Part II

When Peaks Collide Part III

GC Spring Cleaning

Syringes for Gas Chromatography

Extreme Leaks!

 
Tools:

Injection Volume Optimizer / Backflash Calculator

Interactive Polysiloxane Classification Tool

Carrier Gas Flow Rate Calculator

 
CHROMacademy Webcasts / Essential Guides

Basics of split / splitless injection

Optimizing detectors for capillary GC

Column Selection for Capillary GC

Developing Fast GC Separations

Understanding 2D Gas Chromatography

 
 

 

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