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The CHROMacademy Essential Guide Webcast:
Developing Better GC methods - a blueprint

Thursday 19th December 2013, 16:00 GMT

This month we explore what constitutes the optimum GC method. We will examine the many instrument parameter settings, as well as the chemistry and temperature program considerations which can and should be optimized for every analysis.
This webcast analyzes what the ideal method specification might look like and highlights the important decisions which lead to highly stable and sensitive GC methods. The webcast will draw up a blueprint for a highly useful and practically relevant optimum GC method.

 

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Topics covered include:

  • What does the optimum GC method look like?
  • What instrument settings and chemistry choices must be considered in order to design the ideal GC method
  • This webcast highlights all of the decisions required to build a rugged and sensitive GC method
  • All sample, sample inlet, column, temperature programming and detector settings are considered in this highly informative and practically relevant webcast

Who Should Attend:

  • Anyone working with Gas Chromatography who would like to better understand the methods they use
  • Anyone wishing to GC method robustness and sensitivity
  • Anyone involved in developing or transferring GC methods
 


Find out more about this Month's Essential Guide Webcast »

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The CHROMacademy Essential Guide Tutorial
Developing Better GC methods - a blueprint

In order to obtain sensitive, robust, and reproducible gas chromatography (GC) methods, each stage of the chromatographic process needs to be optimized. 
It is also important to record and report as much detail pertaining to each parameter in order for methods to be reproduced between operators and between laboratories.  Using a generic GC method outline, and a generic sample (Table 1), each stage of the GC chromatographic process will be examined resulting in a ‘blue print method’ that can be used to ensure all parameters are considered, optimized and included in the final method specification.

 

Parameter Considerations
Autosampler Syringe, injection speed, sample vials
Column Stationary phase, dimensions
Injection Solvent Matches column polarity
GC Inlet Split, Splitless (Splitless time), Pressure pulsed, inlet temperature
Inlet Liner Shape, deactivation
Injection Volume Backflash
Carrier Gas Type, constant flow/pressure, velocity
Oven Temperature Program Initial Temperature, ramp rate, re-equilibration time
Detector/Detector Gases Gas traps, flow rates, gas ratios

Table 1: Generic GC method

 
 

Syringe

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

Syringe volume
: Choose a syringe so that the injected amount is no smaller than 10% of the syringe nominal volume.1,2  For example, no less than 0.5 μL for a 5 mL syringe and no less than 1 μL for a 10 mL syringe, which 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 (Figure 1).  These syringes contain a needle and a stainless steel fixing that is cemented onto the bottom of the glass syringe barrel; it has 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.

 

Fixed needle syringe.

Figure 1: Fixed needle syringe.

 

Removable needles are recommended for use with experienced operators or where the risk of needle bending is higher (Figure 2).  The syringe has a screw thread or Luer fitting 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; however, they have a lower accuracy and precision.

Removable needle syringe.

Figure 2: Removable needle syringe.

 

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 aspirate samples of normal viscosity.  Medium to high viscosity samples should be diluted or a larger (i.d.) needle may be required.  To reduce the possibility of bending, choose the widest needle OD suitable for the application.  Autosampler syringes with 0.63 mm OD needles should be selected for all applications except on-column injection (Table 2).

 
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 2: Typical needle internal and external diameter combinations.

 

Needle Point Style

The cone shaped needle style has been developed for repeated use with an autosampler 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.  Beveled point needles are recommended for manual injection as they will reduce septum damage in the situation where the septum is not pierced at the exact same point each time.  Dual gauge needles have a narrower tip which is suitable for megabore on-column injections; the wider gauge for the remainder of the needle gives increased strength to the needle for use with autosamplers (Table 3).

 
Tip   Typical Use
Bevel 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 cone Well suited for multi-injection.
Dual gauge dual gauge Suitable for megabore on-column injection.
Dome dome For use with pre-drilled septa.
Side hole side hole Ideal for large volume gas injection.
Sheathed/bevel sheathed bevel Similar to the bevel type but it has stronger mechanical integrity.

Table 3: Typical needle tip styles and uses

 
 

Syringe Washing and Flushing

For manual injection: The syringe should be flushed with approximately 5-10 times its total capacity to eliminate carryover between samples.  This is achieved by repeatedly aspirating and dispensing 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 aspirating sample into the barrel of the syringe and rapidly dispensing the sample whilst keeping the needle tip immersed in the liquid.  Turning the syringe barrel upright, while dispensing the sample may also help remove bubbles.

Autosamplers: Autosamplers will perform pre- and post-injection washes of the syringe/probe.

 

Syringe Care and Maintenance

The integrity of the syringe is of the utmost 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 dichloromethane.  Surfactant materials are also used to clean the barrel.

 

Important:

Do not immerse the 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
  • Re-fit the plunger and flush with acetone
  • Allow syringe to dry
 
 

Injection Speed

The phenomenon of sample discrimination leads to a non-representative sample entering the column.  Figure 3 shows the detector response to an injection of n-alkanes at equal concentration.  The normalized line shows the original sample concentration, and hence, 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 onto the column, furthermore, the recovery of C37 is less than 25%. 

  DiscriminationFigure 3: Discrimination due to differences in boiling point.  Hot split injection of a solution containing equal amounts of n-alkanes in hexane
 
 
 
 

For higher boiling (less volatile) analytes the residence time of the syringe needle in the inlet is too short.  The analyte will condense on the relatively cold inner and outer surfaces of the needle prior to it being withdrawn from the inlet.  Some less volatile analytes may never properly volatilize and the sample passes the split point (head of the capillary column) as a mixture of sample vapor and non-uniform liquid droplets (Figure 4). 

Several approaches to the problem have been postulated:

  • Optimization of liner geometry and packing materials to promote sample mixing and volatilization
  • Optimizing the injection routine (filled needle, hot needle, solvent flush, air flush, sandwich method etc.)
  • High speed autosampler injection (to ensure liquid, rather than vapor injection)
  • PTV injection
  • Cool-on-column injection
  • Improved instrument design to reduce fluctuations in the split flow

In general, the least 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 4: Inlet discrimination »

 

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Vials

Overfilled vials can lead to reproducibility problems due to cavitation (Figure 5).
Cavitation: When a sample is removed from a vial the sample must be replaced with something or a vacuum will occur.  The partial vacuum is not a problem if there is sufficient headspace.
Vials should, therefore, be filled to less than ¾ of its capacity.
With volatile sample diluents cavitation can occur within the needle, where a bubble will form in the needle itself or the connecting tubing.  Vent needles can be used to prevent cavitation.

 

Overfilled sample vial

Figure 5: Overfilled sample vial (left) and correctly filled sample vial (right).

 
 
Parameter Considerations
Autosampler Syringe - Bevel (most common), injection speed = fast,  vials filled ½ - ¾
Column Stationary phase, dimensions
Injection Solvent Matches column polarity
GC Inlet Split, Splitless (Splitless time), Pressure pulsed, inlet temperature
Inlet Liner Shape, deactivation
Injection Volume Backflash
Carrier Gas Type, constant flow/pressure, velocity
Oven Temperature Program Initial Temperature, ramp rate, re-equilibration time
Detector/Detector Gases Gas traps, flow rates, gas ratios
 
 
 

Analytical Chemists

  • I feel empowered to fix things
  • I can troubleshoot effectively
  • I know where to go for help
  • I understand my analyses
  • I know where to get applications
  • I’m up to date
  • I’m more employable
  • My career is progressing
 

Laboratory Managers

  • Improved equipment utilization
  • Faster method development/problem solving
  • Flexible workforce with a common standard
  • Better quality data
  • Get up to speed quicker
  • Lower T&E
  • Less reliant on me
  • I spend less time on training
 

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  • Expert troubleshooting advice when needed
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Stationary Phase

Stationary phases tend to be chosen on the basis of the three main interactions between the analyte and stationary phase; dispersion, dipole, and hydrogen bonding (Table 4).  For example a non-polar analyte will undergo predominantly dispersive type interactions, therefore, for good retention a dispersive type stationary phase should be chosen.

An excellent rule of thumb to help with the choice of stationary phase is ‘like dissolves like’, i.e. the analyte polarity and predominant interaction type should be matched to the stationary phase polarity (Figure 6) and the predominant interaction.

 
Functional Group Dispersion Dipole Hydrogen Bonding
Methyl Strong None None
Phenyl Very strong Weak None
Cyanopropyl Strong Very strong Moderate
Trifluoropropyl Strong Moderate Weak
PEG Strong Strong Moderate

Table 4: Summary of GC stationary phases and their predominant interactions.

 

Polarity

Figure 6: Polarity of GC stationary phases.

 

Stationary phase selection is the most important parameter in GC, however, it can be ambiguous especially when analyzing mixtures of analytes.  For example, the analytes shown in Figure 7 are a homologous series of hydrocarbons, which are a test mix of compounds often used to measure column retention properties.  Their predominant interactions are detailed in Table 5.

 

Homologous series of hydrocarbons


Figure 7:
Homologous series of hydrocarbons

 
Compound Polar Aromatic H-bonding Dipole
Toluene No Yes No Induced
Hexanol Yes No Yes Yes
Phenol Yes Yes Yes Yes
Decane No No No No
Naphthalene No Yes No Induced
Dodecane No No No No

Table 5: Interaction properties of a homologous series of hydrocarbons.
 

From a knowledge of the interaction properties of the analytes there are several suitable column stationary phases that could be chosen including; methyl, phenyl, and cyanopropyl phases.  In fact all of these stationary phase types will separate the analytes; the choice of phase will be determined by a number of factors which give the ultimate selectivity.

Using the ‘like dissolves like’ analogy dispersive phases are typically used to analyze non-polar analytes.  When a series of n-alkanes is chromatographed with a dimethylpolysiloxane (PDMS) column, they elute in boiling point order with the lowest boiling analyte eluting first.  However, the boiling point analogy is lost when a series of aromatic compounds are also chromatographed at the same time (Figure 8).  The elution order (and boiling points) of the homologous series of hydrocarbons used in the separation shown in Figure 8 are detailed in Table 6.  It can be seen that although a PDMS column is often considered as a ‘boiling point column’ the elution order does not hold with the boiling points of these analytes, indicating that the two ‘species’ of analyte undergo different degrees of dispersive interaction with the stationary phase.  It can be seen that the boiling point analogy is often confounded when dealing with analytes with mixed functionality.

 
Elution order Compound Boiling Point (°C)
1 Toluene 111.0
2 Hexanol 158.0
3 Phenol 181.7
4 Decane 174.1
5 Naphthalene 218.0
6 Dodecane 216.2

Table 6: Elution order of a homologous series of hydrocarbons.
 
 

Comparison of the separation of the hydrocarbon sample on a 100% dimethylpolysiloxane (PDMS), 5% diphenyl dimethylpolysiloxane, and 50% diphenyl dimethylpolysiloxane stationary phases are shown in Figure 8.     

All compounds are well resolved on the PDMS and 50% diphenyl dimethylpolysiloxane phase, while co-elution of naphthalene and dodecane is seen on the 5% diphenyl dimethylpolysiloxane phase. 

The phenyl content of the stationary phase may affect retention in a predictable manner.  The higher the phenyl content of the stationary phase, the higher the retention  of aromatic solutes relative to aliphatic solutes – this can be seen in Figure 8 where an increase in phenyl content from 5 to 50% results in greater retention of naphthalene and phenol in comparison to hexanol, decane, and dodecane.  This does not necessarily mean that the aromatics are more retained but that they shift relative to aromatic analytes.  Increasing the phenyl content of the phase will result in earlier elution of n-alkanes.

Polar compounds are more strongly retained by polar stationary phases and vice versa.  An increase in phenyl content of the stationary phase increases the polarity of the column due to the increase in pi electrons.

As a general rule of thumb, if the boiling point of two compounds differs by 30 °C or more then they may be separated by most stationary phases.  This is due to the fact that dispersion is the dominant interaction for a wide range of stationary phases.  If the compounds boiling point differs by less than 10 °C (and the compounds do not belong to a homologous series) then the boiling point analogy is more likely to be in error.

These results show that stationary phase choice can be ambiguous and in many instances there will be a number of columns that may be suitable for analyte separation.  For the purpose of this discussion we will use a 50% diphenyl dimethylpolysiloxane stationary phase

Figure 8: Separation of a homologous series of hydrocarbons

Figure 8: Separation of a homologous series of hydrocarbons using a 100% dimethylpolysiloxane (PDMS) (bottom), 5% diphenyl dimethylpolysiloxane (middle), and 50% diphenyl dimethylpolysiloxane stationary phase (top). 1. Toluene, 2. hexanol, 3. phenol, 4. decane, 5. naphthalene, and 6. dodecane

 
 

Column Dimensions

When choosing a capillary GC column, as well as the stationary phase type, the physical dimensions of the column must also be specified, usually length, internal diameter, and stationary phase film thickness.  All of these dimensions are critical to the performance of any separation.  A useful equation that describes the contributing factors to GC retention time and the ways in which analysis speed can be optimized are shown in Equation 1.

Equation 1

 

 
Length (L)

Column efficiency is proportional to column length.  Doubling column length will double the number of theoretical plates, and hence, double efficiency.  However, resolution is proportional to the square root of efficiency, therefore, doubling column length will only provide a theoretical increase in resolution of a factor of 1.4 (i.e. the square root of 2).  Doubling column length will double analysis time for isothermal operation and increase analysis time by 1.5-1.75 times for gradient temperature programmed analysis.  This therefore, reduces the importance of increasing column length to obtain or improve separation.  In practice column length is only increased when peak separation is very small and high efficiency is required, or when the sample contains many analytes, all of which need to be separated.  Increasing the column length also increases the pressure required to achieve a set flow rate which is not a problem practically unless very narrow columns are used.  Furthermore, column costs will be increased. 

 

Internal Diameter (rc)

Altering the column diameter affects five operational parameters – efficiency, retention, required carrier gas flow rate to achieve optimum linear velocity, capacity, and pressure drop across the column.  The column internal diameter is inversely proportional to column efficiency.  Therefore, halving the column internal diameter doubles the efficiency and improves resolution by a theoretical factor of 1.41.  Increases in efficiency arise due to the increase in analyte/stationary phase interactions in the smaller diameter columns.

Analyte retention is also inversely proportional to the column diameter for isothermal separations, but crucially the retention time change under temperature gradient conditions is 1.25-1.5 times the original retention.  This makes changing the column internal diameter much more attractive in practical terms.

Column head pressure is approximately an inverse square function of column radius – i.e. a 0.25 mm i.d. column requires 1.7 times greater head pressure than a 0.32 mm i.d. column of the same length at the same temperature. 

Column capacity increases with column internal diameter.  The capacity also depends on the stationary phase type, film thickness and the nature of the analytes.  Larger bore columns and thicker stationary phase films have higher analyte capacity and therefore higher analyte masses can be loaded before peak shape begins to deteriorate.

 

Film Thickness (df)
Column stationary phase film thickness (df) affects retention, inertness, capacity, resolution, and column bleed.  Under isothermal conditions, film thickness is directly proportional to retention time (the proportionality is approximately 1.5:1 under temperature gradient conditions).

Thick stationary phase films are used to gain retention for highly volatile analytes such as solvents or some selected permanent gases.  Increasing film thickness allows retention of volatile analytes at temperatures at or above ambient.  Analytes have equal or greater retention at higher column temperatures.  The same principles apply when reducing film thickness, and in this way the retention of highly adsorbed analytes (late eluting, high boiling point, or high molecular weight analytes) may be reduced using thinner film columns.  Doubling film thickness will result in an increase of around 20 °C in elution temperature.

Early eluting analytes (k < 2) are better resolved using thicker film columns.  Resolution will also increase for most analytes with k values between 5 and 10; however, analytes with k > 10 will see no improvement in resolution when a thicker film is used.

For any given stationary phase, thicker films will bleed more and the upper temperature limits of thick film columns will be lower than their thin film counterparts.  Thicker film columns are more inert as the film shields the analyte from active sites on the silica tubing, therefore, increasing film thickness can often improve the peak shape of tailing peaks.  Thicker film columns have a higher capacity and may, therefore, reduce peak broadening; this can be of particular interest when one analyte is present in vast excess compared to the others.  Thicker film columns may prevent co-elution with the larger peak.

 

Phase Ratio (β)
The phase ratio of a column is a measure of the stationary phase to mobile phase ratio at any point in the column and is calculated using Equation 2.

Equation 2

 

Increasing the phase ratio will result in decreased analyte retention.  The phase ratio can be increased by increasing the column radius or decreasing the film thickness.  The opposite is also true; if the phase ratio is decreased analyte retention will increase.  The phase ratio can be decreased by reducing the column internal diameter or increasing the film thickness.  Note that decreasing the phase ratio will result in an increase in column capacity.

The real elegance of using phase ratio is that it can be used to keep retention time approximately constant whilst altering other aspects of the chromatography.  For example, if one wanted to increase the efficiency of a separation this can be achieved by reducing the column internal diameter, however, this leads to increased analysis time at constant pressure and temperature.  However, by choosing a thinner film the phase ratio can be kept approximately constant; the net result is a more efficient separation within the same timescale as the original separation (Figure 9).

 

Effect of altering column dimensions

Figure 9: Effect of altering column dimensions, while maintaining the phase ratio, on a GC separation.  Analysis of a number of GC test compounds of varying functional chemistry, 5% Phenyl polydimethysiloxane, Helium at 35 cm/sec., split injection (1 µl) at a split ratio of 100:1, isothermal at 125 °C
 
 

The phase ratios for common GC column dimensions are detailed in Table 7.

 
Film Thickness df (mm) Column Diameter (mm)
0.1 0.18 0.2 0.25 0.32 0.45 0.53
0.10 250 450 500 625 800 1125 1325
0.18 139 250 278 347 444 625 736
0.25 100 180 200 250 400 450 663
0.40 63 113 125 156 200 281 331
0.42   107 119 149 190 265 315
0.50   90 100 125 160 225 265
0.83     60 75 96 136 160
0.85     59 74 94 133 156
1.00     50 63 80 113 133
1.27       49 63 88 104
1.50       42 53 75 88
2.55       25 31 44 52
3.00       21 27 38 44
5.00       13 16 23 27

 

Table 7: Phase ratio for selected GC column dimensions.

 
 

For the purposes of this discussion the following column will be used:

Parameter Considerations
Autosampler Syringe - Bevel (most common), injection speed = fast,  vials filled ½ - ¾
Column 50%-Phenyl-methylpolysiloxane, 15 m x 0.25 mm i.d., 0.25 μm
Injection Solvent Matches column polarity
GC Inlet Split, Splitless (Splitless time), Pressure pulsed, inlet temperature
Inlet Liner Shape, deactivation
Injection Volume Backflash
Carrier Gas Type, constant flow/pressure, velocity
Oven Temperature Program Initial T, ramp rate, re-equilibration time
Detector/Detector Gases Gas traps, flow rates, gas ratios
 
 
 
 

Analytical Chemists

  • I feel empowered to fix things
  • I can troubleshoot effectively
  • I know where to go for help
  • I understand my analyses
  • I know where to get applications
  • I’m up to date
  • I’m more employable
  • My career is progressing
 

Laboratory Managers

  • Improved equipment utilization
  • Faster method development/problem solving
  • Flexible workforce with a common standard
  • Better quality data
  • Get up to speed quicker
  • Lower T&E
  • Less reliant on me
  • I spend less time on training
 

Subscribe for $399 per/year and access:

  • The entire e-Learning archive
  • All Essential Guide Webcasts and Tutorials
  • LCGCs archive of articles and webcasts
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Stationary Phase

  1. If no information or ideas about which stationary phase to use is available, start with a PDMS or 5%-Phenyl
  2. Low bleed ("ms") columns are usually more inert and have higher temperature limits
  3. Use the least polar stationary phase that provides satisfactory resolution and analysis times. Non-polar stationary phases have superior lifetimes to polar phases
  4. Use a stationary phase with a polarity similar to that of the solutes. This approach works more times than not; however, the best stationary phase is not always found using this technique
  5. If poorly separated solutes possess different dipoles or hydrogen bonding strengths, change to a stationary phase with a different amount (not necessarily more) of the dipole or hydrogen bonding interaction
  6. If possible, avoid using a stationary phase that contains a functionality that generates a large response with a selective detector
  7. 100% Methyl or 5% Phenyl, 50% Phenyl, 14% Cyanopropylphenyl and WAX (PEG) cover the widest range of selectivities with the smallest number of columns
  8. Use PLOT columns for the analysis of gaseous samples at above ambient column temperatures

Column Dimensions

  1. Capillary GC columns are typically 10, 15, 20, 25, 30, 50, 60,120 (m)
  2. Extending the column length is the least favored option for increasing resolution and should be avoided if possible
  3. Cost and analysis time are proportional to column length
  4. Use the shortest column that will give you the required resolution (begin with 25-30 m columns if the number/ nature of samples is unknown).
  5. To increase resolution try changing the stationary phase or column internal diameter first
  6. Narrow internal diameter columns are capable of separating multiple analytes in a single analysis
  7. Increase film thickness when volatile analytes are involved or reduce film thickness to decrease retention of highly adsorbed analytes
  8. Use phase ratio to increase separation efficiency in the same timeframe as the original separation
  9. Column head pressure and bleed increase with column length.
  10. 10-15 m columns are well suited to samples containing well separated analytes or where the number of analytes is low
  11. 50-60 m columns should be used only where very large numbers of components need to be separated and as a last resort when reducing the column internal diameter and changing the stationary phase and temperature program have failed!
 
 

 

A GC sample solvent, usually a lower-boiling point organic solvent, should be capable of dissolving all of the analytes of interest within the sample.  The selection of the solvent should be done by considering not only the injection strategy but also the detection means used for 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 to the analysis of trace levels of higher boiling analytes 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.  The initial temperature of the column should be 20 °C below the boiling point of the sample solvent.

 

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

Solvent polarity should match the polarity of the stationary phase (Figure 10).  This is particularly important in Splitless injection.  A mismatch of solvent in Splitless mode will result in poor peak shape (Figure 11) due to a lack of solvent focusing which can be attributed to the formation of a non-contiguous solvent film.

See GC Sample Introduction eLearning Module / Injection Volume »

 

GC stationary phase and solvent polarities

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Figure 10: GC stationary phase and solvent polarities Figure 11: Splitless injection solvent focusing
 
 

The column that has been chosen for this example method is a 50% diphenyl dimethylpolysiloxane stationary phase, which is a mid-polarity phase, therefore, a mid-polarity solvent should be used, i.e. DCM, CHCl3, t-butyl ether, ethyl acetate etc.

 
Parameter Considerations
Autosampler Syringe - Bevel (most common), injection speed = fast,  vials filled ½ - ¾
Column 50%-Phenyl-methylpolysiloxane, 15 m x 0.25 mm i.d., 0.25 μm
Injection Solvent Ethyl acetate (b.p. 77.1 °C)
GC Inlet Split, Splitless (Splitless time), Pressure pulsed, inlet temperature
Inlet Liner Shape, deactivation
Injection Volume Backflash
Carrier Gas Type, constant flow/pressure, velocity
Oven Temperature Program Initial Temperature, ramp rate, re-equilibration time
Detector/Detector Gases Gas traps, flow rates, gas ratios
 
 
 

Analytical Chemists

  • I feel empowered to fix things
  • I can troubleshoot effectively
  • I know where to go for help
  • I understand my analyses
  • I know where to get applications
  • I’m up to date
  • I’m more employable
  • My career is progressing
 

Laboratory Managers

  • Improved equipment utilization
  • Faster method development/problem solving
  • Flexible workforce with a common standard
  • Better quality data
  • Get up to speed quicker
  • Lower T&E
  • Less reliant on me
  • I spend less time on training
 

Subscribe for $399 per/year and access:

  • The entire e-Learning archive
  • All Essential Guide Webcasts and Tutorials
  • LCGCs archive of articles and webcasts
  • Expert troubleshooting advice when needed
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Split Mode

In split injection mode the sample is introduced into the inlet liner where rapid volatilization occurs.  The sample vapor is then mixed with and diluted by the carrier gas flowing through the center of the liner.  The diluted sample vapor then flows at high velocity past the column entrance where a small portion of it will enter the column.  However, most of the diluted sample will flow past the column entrance and out of the inlet via the split line.  The ratio of column flow to split flow will determine the ratio (or volume fraction) of sample entering the column to that leaving the inlet via the split line.  The split flow rate may be altered to either increase or decrease the amount of sample reaching the column (Figure 12).

Split injection is conventionally used for analyses where the sample concentration is high and the user wishes to reduce the amount of analyte reaching the capillary column.  As capillary columns have a limited sample capacity it is important that the column is not overloaded.  A typical 25 m GC column may contain only 10 mg of stationary phase distributed over its entire length.

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.

 

Figure 12: Split/splitless inlet in split injection mode »

 

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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 vapor that will enter the column (Figure 13).

The calculation of split flow is shown below.  The magnitude of the split ratio will depend on the concentration of the sample injected and the capacity of the capillary column used (Table 8).  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. 

The split ratio is 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 behavior associated with overloading.  Conversely, if the spit ratio is too high too little of the sample will reach the column and the sensitivity will decrease as peak area decreases.

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 ratio should halve the resultant peak area, however, this is not recommended for calibrating the instrument response.  Below a split ratio of around 15:1, further reducing the split ratio may not give a linear relationship.

Figure 13: Setting the split ratio »

 

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Column capacity (in nanograms, Table 8) is the maximum amount of a single analyte that can be loaded onto a column without causing peak shape distortion (fronting peaks are indicative of column overload).  Overloaded peaks will produce problems with integration and may result in co-elution problems.  The column capacities shown in Table 8 are per analyte.

 

Table 8: Capillary GC column capacities. 
ng values are per analyte »

 
Column i.d. Film Thickness
0.1 μm 0.25 μm 0.5 μm 1.0 μm
0.10 mm 10 ng 30-40 ng 50-70 ng 100-200 ng
0.18 mm 20-30 60-80 100-150 250-350
0.25 mm 30-40 125-175 175-250 400-500
0.32 mm 50-70 020-250 250-350 600-800
0.45 mm 80-100 300-400 400-500 800-1000
0.53 mm 100-120 400-500 500-700 1000-1500

 

 

Problems with the Split Line

Most GC instruments are equipped with a filter (usually deactivated charcoal) in the split line to remove potentially harmful species prior to venting to the atmosphere from the split vent.  These filters 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.

 
 

Splitless Injection

Splitless injection is analogous to split injection in many ways.  The hardware used for splitless injection is almost identical to the split injector and most manufacturers will use the same inlet for both split and splitless injection, hence, the name split/splitless inlet.  Just as with split injection the sample is introduced into a hot inlet using a sample syringe where it is rapidly injected and volatilized (Figure 14).  The splitless injector also belongs to the family of vaporizing injectors.  Post injection there are a number of differences in the way that the splitless injector works and a typical splitless injection routine is detailed below.

  • The sample is introduced into the inlet via the septum using a syringe.
  • The sample is vaporized and is mixed with and diluted by the carrier gas.
  • Initially the split line is turned off using a valve to prevent the escape of the sample vapor and carrier gas.
  • ALL OF THE SAMPLE is transferred to the capillary column by the carrier gas during the initial SPLITLESS phase of the injection.
  • The transfer of the sample vapor (diluted with carrier gas) from the inlet is much slower compared to split injection.
  • The sample vapors are trapped (condensed) on the head of the analytical column using a low initial oven temperature.
  • At an optimized time the split line is turned on to clear the inlet of any residual vapors.
  • The oven temperature is programmed to elute the analytes from the column.

The gas flow rate through the liner is equal to the column flow during the splitless phase.  Transfer of the analyte to the column can take minutes which, if no action were taken, would lead to unacceptably wide chromatographic peaks.  Methods for mitigating this will be discussed later in this tutorial.  Splitless injection is typically used for trace and ultra-trace analyses.  The analyte is slowly introduced from the inlet during the whole of the splitless time.  This slow sample vapor transfer would result in the sample band entering the column over a period of 30 – 60 seconds.

 

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Figure 14: Split/splitless inlet in splitless injection mode

 
 

Optimizing Splitless time

During splitless injection it is of vital importance that the inlet is purged of residual vapors 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 to the capillary column. 

The inlet purge is achieved by actuating the split (purge) valve which allows a high split flow through the liner which quickly purges the residual vapors 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.

It is vital that the splitless time is optimized for each application.  Too short a splitless time will mean that the analyte still resident in the liner will be discarded via the split line.  This may lead to poor analytical sensitivity and reproducibility (Figure 15).

Too long a splitless time will lead to badly tailing solvent peaks, extraneous peaks, and a rising baseline making reproducible integration difficult (Figure 16).

 
Splitless time too short   Splitless time too long
     
Figure 15: Splitless time too short – loss of higher boiling analytes   Figure 16: Splitless time too long – loss of higher boiling analytes
 
 

The splitless time is usually empirically optimized by monitoring the peak area of an early, mid, and late eluting peak in the chromatogram.  The peak area is plotted against the splitless time (Figure 17).  For reproducible analysis the splitless time should be chosen just in the plateau of the area response curve as indicated.  Typical splitless times lie in the region 20- 90 seconds.

 

Figure 17: Area response curve used to optimize splitless time »

  Area response curve
 
 

Pressure Pulsed Injection

A technique known as pressure puled injection may be used to minimize the risk of backflash.  An initial pressure pulse is applied to the inlet while the injection is being made and the gas is expanding.  The increased head pressure restricts the gas expansion stopping backflash.  The sample is then rapidly transferred through the injector and on to the column at the same time as compressing the sample band.  The column flow is increased during the pressure pulse which would reduce the split ratio (if split injection is used) and this will increase sensitivity (Figure 18).

Figure 18: Example of a pressure pulsed injection »

  pressure pulsed injection
 
 
 
 

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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 volatilization of all sample components.  This will ensure that temperature (inlet) discrimination of higher boiling (less volatile) analytes is minimized. 

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

The accepted arbitrary inlet temperature to begin method development for new analytes and applications is 250 °C.  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. particularly when thermally labile analytes are being analyzed or where the sample is high boiling).

With the inlet set to 250 °C a scouting temperature gradient can be employed which will elute analytes and sample components over a wide range of boiling points (Figure 19).  This allows for the elution temperature of the highest boiling component to be determined.  The inlet temperature should then be set to at least 50 °C above this temperature to ensure efficient volatilization.

 
GC temperature scouting gradient  

« Figure 19: GC temperature scouting gradient.

 

 

 

For the generic method a splitless injection will be used, with a splitless time of 50 s, and an inlet temperature of 268 °C which corresponds to 50 °C above the boiling point of the highest boiling analyte in the analyte mixture (naphthalene 218 °C).

 

 
 
Parameter Considerations
Autosampler Syringe - Bevel (most common), injection speed = fast,  vials filled ½ - ¾
Column 50%-Phenyl-methylpolysiloxane, 15 m x 0.25 mm i.d., 0.25 μm
Injection Solvent Ethyl acetate (b.p. 77.1 °C)
GC Inlet Splitless (50 s), 268 °C
Inlet Liner Shape, deactivation
Injection Volume Backflash
Carrier Gas Type, constant flow/pressure, velocity
Oven Temperature Program Initial Temperature, ramp rate, re-equilibration time
Detector/Detector Gases Gas traps, flow rates, gas ratios
 
 

 

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 volatilized 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 vapors 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 20 outlines the main features of typical GC inlet liners for a better understanding.  The main variables in GC liner selection are:

  • Liner internal diameter
  • Packed/unpacked
  • Packing position
  • Liner internal geometry
  • Upper gooseneck
  • Lower gooseneck
  • Inverted (Jennings) cup
  • Baffled
  • Deactivation type
 

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Inlet Liners for Split Injection

Split injection is a fast way to introduce a sample on to a GC column.  This injection mode is well suited for both highly concentrated and/or dirty samples.  Due to the short inlet residence time split liners (Figure 21) are designed in such a way that sample vaporization is maximized.  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.

 

GC liner designs


Figure 21: GC liner designs for split injection.

 
 
 

Inlet Liners for Splitless Injection
Useful for low concentration samples, this technique involves an initial hold time.  Therefore, liners for splitless injection do not require high surface area for sample vaporization (Figure 22).

 

Figure 22: GC liner designs for split injection »

 

  GC liner designs for split injection
 
 

The selection of an incorrect liner can result in a myriad of problems.  Not only inaccurate quantification but sample backflash, peak tailing, irreversible adsorption, and mass discrimination issues are not uncommon with poor liner choice (Figure 23).  A good liner will:

  • Minimize mass discrimination by ensuring complete sample vaporization.
  • Have a larger volume than the total volume of vaporized sample and solvent.
  • Not react with the sample (deactivated)

Figure 23: Peak shape issues resulting from the wrong choice of liner internal diameter »

  Peak shape issues

 

The addition of quartz wool will increase the vaporization surface area while promoting mixing of the sample and carrier gas.  Furthermore, the use of quartz wool will reduce the incidence of particulate matter entering 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 becomes dirty.  It should be exchanged on a regular basis to prevent chromatographic problems.  Avoid the use of glass wool when it is not advantageous.

By using liners packed with selective adsorption material such as Tenax®, Carbotrap®, or Chromsorb®, the range of components that can be trapped in the liner can be significantly extended towards the more volatile components (Figure 24).  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.

 

Large volume injection of a 30 ppm solution of normal alkanes

Figure 24: Large volume injection of a 30 ppm solution of normal alkanes.  Liner packed with a) Tenax® and b) glass wool.

 
 

If the packing material is placed near the top of the inlet it will wipe any sample components from the syringe needle, it can improve injection precision, and help prevent backflash.  Placed near the bottom of the liner the packing material will aid the vaporization of high molecular weight compounds, and help to increase mixing of the sample vapor with the carrier gas.
Smaller liner i.d. results in sharper peaks as there is less time available for diffusion of the sample plug within the liner (Figure 25).  If analyte boiling points are close or if there are only small differences in functional group chemistry a narrow i.d. liner should be used. 

Remember: Consider backflash when using a smaller i.d. liner.

 

Figure 25: Peak width resulting from an injection of 0.1 μL of acetone in liners of differing internal diameter »

  Peak width
 

For the example method discussed here a splitless injection is being used, therefore, a gooseneck liner has been chosen.  The dimensions and a part number are useful to include in the method as this provides the information that is useful when calculating the correct injection volume and if the liner needs replacing.   

 
Parameter Considerations
Autosampler Syringe - Bevel (most common), injection speed = fast,  vials filled ½ - ¾
Column 50%-Phenyl-methylpolysiloxane, 15 m x 0.25 mm i.d., 0.25 μm
Injection Solvent Ethyl acetate (b.p. 77.1 °C)
GC Inlet Splitless (50 s), 268 °C
Inlet Liner Gooseneck, (7.5 mm x 4 mm i.d., P/N is useful), fused silica wool packing, deactivated
Injection Volume Backflash
Carrier Gas Type, constant flow/pressure, velocity
Oven Temperature Program Initial Temperature, ramp rate, re-equilibration time
Detector/Detector Gases Gas traps, flow rates, gas ratios
 
 
 
 
 

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The volume of the sample solvent injected into the split/splitless inlet will have a major effect on the accuracy and reproducibility of the quantitative analysis and the chromatographic peak shape.  As the injection is being made, the sample solvent rapidly volatilizes 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 vapor (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 from this injection will again backflash into the gas lines.  In this instance the analyte deposited during the previous injection will be lapped back into the inlet, ultimately finding its way onto the column.  This will cause carryover and will reduce quantitative accuracy and reproducibility (Figure 26).

 

Figure 26: Backflash »

 

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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 the particular set of temperature and pressure conditions (Figure 27).  The risk of backflash can be mitigated by using a smaller injection volume, increasing the head pressure or using pressure pulsed injection (Table 9).

Figure 27: Backflash Calculator »

 

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Solvent Vapor Volume
1 μL injection 0.5 μL injection
Hexane 195.68 97.84
Toluene 240.06 120.03
Ethyl Acetate 259.31 126.65
Methanol 630.83 315.41
Water 1413.33 706.67
 

« Table 9: Calculated vapor volumes for GC solvents.  Liner length: 75 mm, liner i.d.: 4 mm, head pressure: 10 psi, injection port temperature: 250 °C.

 

The solvent used in our example GC method is ethyl acetate.  Table 9 shows that the volume of vapor generated in a 1 and 0.5 μL injection is 259.31 and 126.65 μL, both of which are less than the volume of the liner being used (900 μL), therefore, either injection volume would not produce a risk of backflash. 

 
 
Parameter Considerations
Autosampler Syringe - Bevel (most common), injection speed = fast,  vials filled ½ - ¾
Column 50%-Phenyl-methylpolysiloxane,
15 m x 0.25 mm i.d., 0.25 μm
Injection Solvent Ethyl acetate (b.p. 77.1 °C)
GC Inlet Splitless (50 s), 268 °C
Inlet Liner Gooseneck, (7.5 mm x 4 mm i.d., P/N is useful), fused silica wool packing, deactivated
Injection Volume 1 μL
Carrier Gas Type, constant flow/pressure, velocity
Oven Temperature Program Initial Temperature, ramp rate, re-equilibration time
Detector/Detector Gases Gas traps, flow rates, gas ratios
 
 
 
 

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The type of carrier gas used for a separation has a significant practical impact.  Practically speaking a balance between efficiency and analysis time is required for each GC method.  The van Deemter plot (Figure 28) shows the optimum practical gas velocity (OPGV) working regions of various GC carrier gases. 

Using nitrogen will give the most efficient separation (it has the lowest value HETP minima) as it is the most viscous and analyte longitudinal diffusion is minimized.  However, the minimum point on the curve corresponds to a relatively low linear velocity (Table 10).  Whilst this would be suitable when using packed GC columns with large internal diameter, it would result in unacceptably long run times with capillary columns.

Hydrogen and helium carrier gases both show optimum efficiency at higher linear velocity, which will produce shorter analysis times than those achieved with nitrogen while generating almost comparable efficiency.  A further significant advantage of using hydrogen is the flatness of the van Deemter curve to the right of the minimum.  This indicates that significantly higher linear velocity can be used without compromising separation efficiency.

 

Van Deemter plot


Figure 28: The optimum practical gas velocity working regions of various GC carrier gases. Note that the hydrogen OPGV (solid lines, within 25% of the plate height minimum) can range up to 100 cm/s depending upon application type and column dimension.

 
 
Carrier Gas Optimum Linear Velocity (cm/sec)
H2 30-50
N2 10-15
He 20-40

 

« Table 10: Values for optimum practical gas velocities (OPGV) for GC carrier gases.
 
 

Linear velocity should be optimized for each separation and is dependent upon the carrier gas and column dimensions (Equation 4).

 
 

Relative viscosity of common GC carrier gases Relative Gas Velocities and Diffusivity: Effect on Inlet Pressure and Liner Selection

Undoubtedly two properties of a gas play a major role in the GC process: diffusivity and viscosity.  The diffusivity of nitrogen and helium are roughly the same but hydrogen is a bit less than half as viscous as helium at the same temperature (Figure 29).  For this reason hydrogen requires a lower pressure to achieve the same average carrier gas velocity as for helium.

 

 

Figure 29:  Relative viscosity of common
GC carrier gases at various temperatures
»

 
 

Column inner diameters With EPC systems, choosing the correct gas type and required linear velocity results in an automatic inlet pressure adjustment.  Some column dimension/flow rate combinations will lead to very low inlet pressure requirements (Figure 30). 

 

Figure 30: Column inner diameters (mm): (a) 0.20, (b) 0.25, (c) 0.32, (d) 0.53; column temperature: 50 °C; average linear velocity: 40cm/s »

 

 
 

This can be particularly problematic with split injection (>10:1 split) due to the increase in inlet head pressure that occurs when the split flow or split ratio is increased (Figure 31).  Issues with pressure stability and retention time reproducibility may result.

 

Figure 31: Increasing the split flow or split ratio increases the inlet head pressure, reduces sample transfer time and reduces diffusion of the resulting gaseous sample »

 

Some practical points to remember that will help when changing carrier gas depending on the sample injection type you are using are:

  1. Split Injection - When changing from helium to hydrogen it might be necessary not only to adjust the split ratio.  Method translation may mean a change in column flow rate.
  2. Splitless Injection - Hydrogen is preferred over helium as it carries the solute from the inlet into the column faster, so sharper peaks are obtained.  This allows for lower detection limits.
  3. Direct Injection - There is no concern on the conversion when using either hydrogen or helium.
 

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Constant Flow or Constant Pressure

In a chromatographic system at constant pressure an increase in gas viscosity results in a decrease in the linear velocity (and flow) of the carrier gas, ultimately resulting in lower column efficiency and unacceptably long retention times for more highly retained analytes (Figure 32).  The increase in gas viscosity is due to the change in temperature during GC analysis (Figure 33). 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 which will lead to a steadily rising or falling baseline.  This can make integration of the peaks in the chromatogram difficult and irreproducible.

Using electronic pressure control (EPC) 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.

 
 

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Figure 32: Examples of GC separations carried out under constant pressure and constant flow. 

 

Figure 33: Effect of temperature on gas viscosity

 
 
Parameter Considerations
Autosampler Syringe - Bevel (most common), injection speed = fast,  vials filled ½ - ¾
Column 50%-Phenyl-methylpolysiloxane, 15 m x 0.25 mm i.d., 0.25 μm
Injection Solvent Ethyl acetate (b.p. 77.1 °C)
GC Inlet Splitless (50 s), 268 °C
Inlet Liner Gooseneck, (7.5 mm x 4 mm i.d., P/N is useful), fused silica wool packing, deactivated
Injection Volume 1 μL
Carrier Gas H2, constant flow or constant pressure 40 cm/sec
Oven Temperature Program Initial Temperature, ramp rate, re-equilibration time
Detector/Detector Gases Gas traps, flow rates, gas ratios
 
 
 
 

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Initial Temperature and Hold Time

The initial temperature and hold time generally affect the resolution of early eluting peaks within the chromatogram, with little or no effect on late eluting peaks.  Adjusting the initial temperature has a greater effect on the resolution of early eluting peaks than adjusting the hold time (Figure 34).

Decreasing the initial temperature will improve the resolution between early eluting peaks if required, however, the overall analysis time will be increased due to longer run and cool down times.  If the first peaks in the chromatogram do not elute for a significant time the initial temperature can be increased to save time.  Care should be taken not to compromise resolution of early eluting peaks.

Lowering the initial hold time can sometimes increase resolution between early eluting peaks without increasing analysis time, however, the effects are less noticeable than with initial temperature.  Conversely, shortening the initial hold time can greatly improve peak shape and efficiency, sometimes also improving the resolution of early eluting peaks.  It should be noted that extending the initial hold time past about 5 minutes can often lead to broad peaks and loss of resolution due to decreased efficiency.

Some good rules of thumb for choosing an initial oven temperature and hold time when using split/splitless injection are as follows:

  • Lowest practical GC oven temperature is 40 °C (without using cryogenics)
  • Split injection
    • Normally no initial hold time is required
    • If lower oven temperatures are used (retention of volatile analytes) an initial hold may be needed but should be kept as short as possible
    • If the oven temperature is < 30 °C less than solvent B.P. use a hold time 
    • Start with 30 sec. and increase if necessary

  • Splitless injection

  • Golden Rule
    Initial oven temperature should be 20 oC < solvent B.P.

     

  • Requires an initial hold time to cryo and solvent focus the sample
  • Match hold time to the splitless time
 

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  « Figure 34: Setting initial oven temperature and hold time
 
 

Ramp Rate and Mid-Ramp Holds

Adjusting the rate at which the temperature increases during the thermal gradient (ramp rate) has the most pronounced effect on the resolution of analytes which elute in the middle of the chromatogram.

We are often asked here at CHROMacademy what the optimum ramp rate is and the best advice we have ever been given was by the famous chromatographer Walt Jennings who once said that the optimum ramp rate (in °C/min) is equal to 10 °C per void time (t0) (Equation 4).  Void time is equal to the retention time of an unretained compound.

 

Once the optimum ramp rate for your system has been set it can be further optimized if required using ±5 °C/min. steps.  If higher or lower steps are used drastic or insignificant (respectively) changes may occur.

If there are a critical pair of peaks that cannot be resolved by altering or using multiple ramp rates follow the steps below.

  1. Determine the elution temperature of the critical pair.
  2. Use Giddings approximation (Equation 5) to determine the temperature at which to include an isocratic hold, i.e. subtract 45 °C from the temperature at which the critical pair elutes.
  3. Start with a 1 minute hold and increase until separation occurs.

For the example method discussed here an initial oven temperature of 57 °C is set as this follows the Golden Rule of Splitless injection, which states that the initial oven temperature should be 20 °C below the solvent boiling point (ethyl acetate 77.1 °C).  As a splitless injection is being performed the initial hold should match the Splitless time, hence, an initial hold of 50 s is programmed into this method.  An optimum ramp rate should be considered for each separation, for example, 20 °C for our method.  Finally, if there are no analytes left to elute the final temperature should be set 10 – 30 °C above the elution temperature of the final analyte (Equation 6).



Where:
Ti = initial temperature (°C)
tf = elution time of the final analyte

 

 
 
Parameter Considerations
Autosampler Syringe - Bevel (most common), injection speed = fast,  vials filled ½ - ¾
Column 50% - Phenyl-methylpolysiloxane, 15 m x 0.25 mm i.d., 0.25 μm
Injection Solvent Ethyl acetate (b.p. 77.1 °C)
GC Inlet Splitless (50 s), 268 °C
Inlet Liner Gooseneck, (7.5 mm x 4 mm i.d., P/N is useful), fused silica wool packing, deactivated
Injection Volume 1 μL
Carrier Gas H2, constant flow or constant pressure 40 cm/sec
Oven Temperature Program 57 °C (hold 50 s) , 20 °C/min to 270 °C
Detector/Detector Gases Gas traps, flow rates, gas ratios
 
 
 
 

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Flame Ionization Detector (FID)

The flame ionization detector is the most widely used GC detector (Figure 35).  Its high sensitivity and linear range for carbon containing compounds make it very popular in organic analysis.  The flame ionization detector produces a proportional response to the number of carbon atoms in a molecule.

 

Figure 35: Flame ionization detector »

 

Since water is a product of combustion the FID detector should be maintained at temperatures above 125 °C to prevent condensation of water and high boiling point sample species.  Typically most FID detectors will operate at or above 250 °C, depending upon the application.  Temperature does affect sensitivity of the detector although in practice the detector temperature is rarely a parameter that is important to optimize.  If required start with a detector temperature of 250 °C and increase and decrease the temperature in 20 °C steps to investigate the temperature/sensitivity relationship for particular analytes.

 

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Foe efficient operation the ratio (stoichiometry) of the gases used must be correct (Figure 36).  This is usually in the order of 1 carrier : 1 fuel (hydrogen) : 10 oxidizer (air).  For capillary applications the flow rates (in mL/min) are usually in the ratio of 30 : 30 : 300-400 mL/min.  For capillary column applications where the carrier gas flow rate is less than 30 mL/min a make-up gas is used to increase the column effluent volume; this serves to dilute the effluent stream  (to keep within the detector linear range) and propels the analyte up into the body of the flame.  The make-up gas is usually chosen to be different from the carrier, with nitrogen being the most popular as its viscosity ensures good mixing with the effluent stream.

 

Figure 36: Optimization of gas stoichiometry for a FID detector »

 

 

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Gases and gas traps 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 contaminants of concern are oxygen, moisture, and hydrocarbons.  These impurities can be removed using gas traps which should be fitted as close to the GC instrument as possible.  The correct gas traps must be installed in the correct order.  For a GC system with an FID detector the correct gas traps, and the order in which they should be installed, are shown in Figure 37.

 

« Figure 37: Gases and gas traps (in the order they should be installed) that are required for a GC system containing a flame ionization detector (FID) where the make-up gas is the same as the carrier (top) and the make-up gas is different to the carrier (bottom). 

 
 
 

Nitrogen Phosphorous Detector (NPD)

Although the nitrogen phosphorous detector (NPD) is based on a similar design to the FID and belongs to the family of ionizing detectors it works on a different principle to the FID (Figure 38).  It has enhanced sensitivity and selectivity towards compounds containing nitrogen and/or phosphorous, therefore, it finds favor in applications where analytes of this type are being analyzed.

The voltage applied to the bead, and hence the resulting temperature, does have a marked effect on the detector response.  If unsure start at 2 V and make 10 mV adjustments until the optimum response is obtained.  It is essential to ensure that the hydrogen flow rate is low enough NOT to sustain a flame at the jet tip otherwise measurement of nitrogen containing compounds will not be possible.  The detector is sensitive to variations in the hydrogen flow rate and a constant flow of hydrogen is recommended to ensure steady baselines.  Nitrogen is preferred over helium as a make-up gas due to its lower thermal conductivity.  Using nitrogen the source requires a lower heating current.  The detector is mass-flow sensitive and as such it is better to operate in constant flow as opposed to constant pressure mode when performing temperature programmed analysis. 

To increase bead lifetime the following steps should be followed:

  • Use the lowest practical bead voltage
  • Run clean samples
  • Turn the bead off when not in use
  • Keep the detector temperature high (320 to 335 °C)
  • Turn the hydrogen flow off during solvent peaks and between runs
  • If your NPD is off for a long time in a high-humidity environment, water may accumulate in your detector.  To evaporate this water:
    • Set the detector temperature at 100 °C and maintain it for 30 minutes
    • Set the detector temperature to 150 °C and maintain it for another 30 minutes
 

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Figure 38: Nitrogen phosphorous detector

 
 

The NPD detector is extremely sensitive to hydrocarbon impurities in the hydrogen and air supply for the flame.  Hydrocarbon impurities may result in increased baseline noise and reduced detector sensitivity.  Water and oxygen can react with the GC column stationary phase resulting in column bleed which in turn reduces analyzer sensitivity, increased baseline noise, and decreased column lifetime.  The gases and gas traps (in the order they should be installed) for a GC system containing a NPD detector are shown in Figure 39.

 

Gases and gas traps

 

« Figure 39: Gases and gas traps (and the order in which they should be installed) that are required for a GC system containing a nitrogen phosphorous detector (NPD).

 

 
 

Electron Capture Detector (ECD)
The electron capture detector (ECD) measures the electrical conductivity of the effluent gas stream resulting from exposure to ionizing radiation from a radionuclide (Figure 40).  It is a selective detector that responds to compounds that are capable of capturing electrons, especially halogenated compounds.

When the detector is used with a capillary column a make-up gas is generally required, it is convenient to use less expensive, but high quality, nitrogen as the make-up gas and the more expensive helium as the carrier gas.

Improved performance and linearity can be obtained by operating the detector in a pulsed mode.  A square wave pulse (amplitude 50 V, width 1 ms at intervals of 20-50 μs) is applied at a frequency that maintains a constant current in the detector cell.  In order to maintain the current in the presence of an analyte the pulse frequency has to be increased.  The signal is generated in proportion to the frequency of the applied pulse.

The cleanliness of the detector needs to be maintained at all times which often means care needs to be taken with sample preparation.  Chromatographic peaks obtained with a dirty ECD have a distinctive shape and the sensitivity of the detector can often increase as the detector performance deteriorates.

 

Figure 40: Electron capture detector »

 

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The carrier gases used for ECD operation should be pure and dry.  Oxygen and water are both electronegative and as such contribute to noisy baselines.  Gases and gas traps (and the order in which they should be installed) that are required for a GC system containing an electron capture detector (ECD) are shown in Figure 41.

 

Gases and gas traps

 

 

« Figure 41: Gases and gas traps (and the order in which they should be installed) that are required for a GC system containing an electron capture detector (ECD).

 

 
 

Mass Spectrometer – Selective Ion monitoring (SIM)
In selective ion monitoring (SIM) mode the MS gathers data for masses of interest rather than looking for all masses over a wide range.  This allows selective analysis of a particular analyte of interest.  Typically 2-4 ions will be monitored per compound; the ratio of these ions will be unique to the specific analyte.  Sensitivity can be increased by optimizing the mass scan rate and dwell time (time spent looking at each mass).  GC/MS-SIM can increase sensitivity by 10-100 (c.f. GC/MS-Full scan).

In order to maximize instrument sensitivity the roughing pump oil should be well ballasted to achieve the bets vacuum levels.  The source should be tuned with tuning compounds with ions close to those of the analytes of interest to enhance sensitivity.

 

In summary, we have examined each of the parameters that should be included in, and optimized, for every GC method resulting in a ‘blueprint’ method.  It is important to consider each of these parameters as this will lead to more sensitive and robust chromatography.  It is also pertinent to fully report each chromatographic parameter in order to maintain a precise record of the analysis.

We have shown that one of the most important parameters to consider when developing or improving a GC method is the column; including the stationary phase and column dimensions.  Some excellent rules of thumb to aid in the decision of column stationary phase is ‘like dissolves like’, in other words match the column polarity to that of the analytes.  In regard to column dimensions a 25-30 m column is a good starting length, and changing the column diameter or stationary phase is a better solution to improve resolution than altering the column length.
Injection solvent is of the utmost importance when using Splitless injection which requires solvent focusing to give efficient chromatographic peaks, and should be matched with the column polarity.

The mode of inlet operation should be chosen depending on the type of analysis being carried out, i.e. split injection for highly concentrated samples and splitless injection for trace analyses.  The inlet temperature can be optimized using scouting gradients and should ensure efficient volatilization of all analytes of interest.  Finally, in splitless mode the splitless time must be optimized to ensure that all analytes are transferred to the column and the resulting chromatography produces good peak shapes.

Injection volume should be optimized for each analysis to avoid the risk of backflash which will result in ghost peaks in subsequent chromatographic runs.

The inlet liner is an important consumable and is available in a wide range of designs that are appropriate for each type of injection mode.  Smaller diameter liners can result in sharper peaks or can be used if analyte boiling points are close or if there are only small differences in functional group chemistry.  Packing material or liners with tortuous paths can be utilized to aid volatilization of high boiling point analytes.  By using liners packed with selective adsorption material such as Tenax®, Carbotrap®, or Chromsorb®, the range of components that can be trapped in the liner can be significantly extended towards more volatile components.
The type of carrier gas used for a separation has a significant impact.  Practically speaking a balance between efficiency and analysis time is required for each GC method and linear velocity should be optimized for each separation and is dependent upon the carrier gas and column dimensions.

We have shown when to use an initial hold time and how to optimize this parameter and demonstrated that it is often better to reduce the initial oven temperature instead of including an initial hold time.  However, splitless injection will always require an initial hold time in order to cryo focus the sample band.  If there are critical pairs the Giddings approximation can be used to estimate where mid ramp holds should be used to obtain resolution.  Finally, adjusting the rate at which the temperature increases during the thermal gradient (ramp rate) has the most pronounced effect on the resolution of analytes which elute in the middle of the chromatogram.  The optimal ramp rate for a separation is 10 °C per void time (t0). 

We have discussed the operating parameters for each of the major detectors used in GC and how to optimize each detector to obtain the maximum sensitivity.  The use of gas filters, the type of filter, and how the filters should be installed has also been outlined.

 
 

GC blueprint

 

 

 
 
 
 
 
 
 
 

 

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The Essential Guide from LCGC’s CHROMacademy presents the definitive guide in translating your GC and GCMS methods.  In this session, Jaap De Zueew (Restek Corporation) and Tony Taylor (Technical Director, Crawford Scientific) explore what constitutes the perfect GC method.  Whilst a perfect method does not exist – many of the instrument parameter settings, chemistry and temperature program considerations can be optimized for the analysis at hand.  This webcast analyses what the perfect method specification might look like, and highlights the important decisions which lead to highly stable and sensitive GC methods.  The webcast will draw up a blueprint for a highly useful and practically relevant ‘perfect’ GC method...

Jaap de Zeeuw
International GC Consumables Specialist, Restek Corporation

Tony Taylor
Technical Director
Crawford Scientific

Key Learning Objectives:

  • Identify key parameters which affect sensitivity and robustness in GC methods
  • Examine key sample and inlet conditions which affect peak shape and quantitative accuracy
  • Highlight key choices with stationary phase selection and column geometry
  • How to build robust and effective temperature programs
  • Optimizing detector response for a range of common GC detectors
  • Building in quantitative robustness to GC methods
  • Highlight the key variable for consideration when developing or optimizing GC methods and using these to build a GC Method blueprint
  • Highlight the minor as well as the major instrument variables which affect GC method quality