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The CHROMacademy Essential Guide Webcast:
Understanding GC Methods.

Thursday, October 24th 2013, 16:00 GMT.

The Essential Guide from LCGC’s CHROMacademy presents the first in a series of webcasts aimed to develop the skills and understanding of GC users.  In this session, Dr. John Hinshaw (Senior Scientist, BPL Global Ltd) and Tony Taylor (Technical Director, Crawford Scientific) take an in-depth look at principles of GC sample introduction and the associated variables which need to be defined in GC methods.  We describe the principles of sample injection and describe in detail how method variables are set and optimized using real life ‘good’ and ‘bad’ method examples.

  • A series of in-depth webcasts designed to improve your GC and GC-MS skills
  • Understand the critical variables in GC and GC-MS methods
  • Learn how to set major and minor method variables
  • Examples of good and bad methods – recognizing when problems might occur and improve your troubleshooting skills
  • Lots of tips and tricks from the chromatographers toolbox
  • Part 1 – Sample Introduction Variables
 

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Who Should Attend:

  • Anyone using GC who would like to improve their understanding of the technique
  • Anyone who needs to understand GC methods and the implications of major and minor sample introduction variables on method performance
   


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The CHROMacademy Essential Guide Tutorial
Understanding GC Methods

The Essential Guide from LCGC’s CHROMacademy presents the first in a series of webcasts aimed to develop the skills and understanding of GC users.

In this session, we take an in-depth look at principles of GC sample introduction and the associated variables which need to be defined in GC methods. 

We describe the principles of sample injection and describe in detail how method variables are set and optimized using real life ‘good’ and ‘bad’ method examples.

  • Understand the working principles of the Split / Splitless Injector
  • Appreciate the major and minor variables which control sample injection
  • Recognize when a method is deficient in terms of the amount of information given and make informed choices about the parameters which are not stipulated
  • Appreciate the importance of proper inlet function and make informed choices in terms of inlet liners
  • Understand how to set and optimize sample injection variables based on sample (analyte) type and intended analytical outcomes

 

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The most common inlet for capillary GC is known as the Split/Splitless inlet which, as the name suggests, can be operated in two modes, split or splitless (Figure 1).  The inlet belongs to a broad range of inlet types known as vapourising inlets as during the injection process the liquid sample is vapourised into the gas phase prior to transfer onto the capillary column.  The Injection principle of the split/splitless inlet is outlined below:

  • A syringe containing the sample is used to pierce a rubber septum
  • The sample is rapidly introduced into the heated inlet
  • The sample liquid rapidly volatilises to the gaseous form and is constrained within a glass liner of fixed volume
  • The gaseous sample is swept onto the column by the carrier gas
  • Depending upon the mode of operation some of the sample may be directed away from the column
 

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Figure 1: Split/splitless inlet.

 

The gas flow paths in the split/splitless inlet has one point of entry (the inlet gas supply) and three exit points from the inlet (septum purge, split line, and capillary column).


Inlet gas supply

The gas flow (pressure) necessary to maintain the correct flows at the various outlets is supplied here.  Typically a backpressure regulated supply, the ‘total flow’ as it is sometimes known will supply the flows used for:

  • Capillary column flow (carrier gas flow)
  • Split flow (for split injection mode)
  • Septum purge flow

The gas used will be the carrier gas used for the GC separation, typically hydrogen, helium, or nitrogen.

 
 

Septum purge gas outlet

Septa are manufactured from rubber or polymeric composite materials containing plasticisers and other compounds that are capable of outgassing at elevated temperatures.  In order to reduce the number of these contaminant compounds reaching the detector or fouling the GC column the septum purge gas flows across the underside of the septum and flushes the outgassing products to waste via the septum purge gas outlet.


The septum purge gas also helps to prevent contamination of the underside of the septum by the sample components that may overspill from the inlet liner during injection.  This will help reduce carryover from injection to injection.
The septum purge flow rate is typically regulated using a forward pressure valve or pneumatic regulator and typical flow rates are in the region 2-5 mL/min.


Split valve/split line gas outlet

This arrangement is used to discard some or all of the liner contents.  In split injection the valve is constantly in the open position and the gas flow is regulate to determine the fraction of the sample that is discarded relative to that which reaches the capillary column.


In splitless injection the valve is initially closed to ensure all of the analyte is transferred to the capillary column.  It is then opened after an optimised period of time to discard residual solvent and sample vapours.

 
 

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In split injection mode the sample is introduced into the inlet liner where rapid volatilisation occurs.  The sample vapour is then mixed with and diluted by the carrier gas flowing through the centre of the liner.  The diluted sample vapour 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.
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 volatilised 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 that the boiling point of the sample solvent.

 

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Figure 2: Split/splitless inlet in split injection mode.

 
 

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 (Figure 3).


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 1).  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 behaviour 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 reducing the split ratio may not give a linear relationship.

 

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Figure 3: Setting the split ratio.

 

Column capacity (in nanograms, Table 1) 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 1 are per analyte. 

 

Table 1: Capillary GC column capacities.  ng values are per analyte.

 
 

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 (Figure 4).  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.

 

Figure 4: Split vent trap.

 
 

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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 volatilised (Figure 5).  The splitless injector also belongs to the family of vapourising 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 vapourised 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 vapour 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 vapour (diluted with carrier gas) from the inlet is much slower compared to split injection
  • The sample vapours are trapped (condensed) on the head of the analytical column using a low initial oven temperature
  • At an optimised time the split line is turned on to clear the inlet of any residual vapours
  • The oven temperature I programmed to elute the analytes from the column
 
 

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Figure 5: Split/splitless inlet in splitless injection mode.

 
 

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 unacceptable 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 vapour transfer would result I a band entering the column over a period of 30 – 60 seconds.
For example the conditions shown in Table 2 can be used to calculate the transfer time of the analyte in a splitless injection, 28 seconds for this example (Equation 1).

 
Inlet liner   Deactivated double taper liner (V = 800 μL = 0.8 mL)
Injection volume   0.5 μL
Injection solvent   Acetonitrile
Head pressure   3.8 psi
Carrier   He, Constant flow 1.7 mL/min

Table 2: Example splitless GC method.

 
 
 

Optimising Splitless time

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 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 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.


It is vital that the splitless time is optimised 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 6).

 

Figure 6: Splitless time too short – loss of higher boiling analytes.

 
 
Too long a splitless time will lead to badly tailing solvent peaks, extraneous peaks, and a rising baseline making reproducible integration difficult (Figure 7).
 

Figure 7: Splitless time too long – loss of higher boiling analytes.

 
 

The splitless time is usually empirically optimised 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 8).  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 8: Area response curve used to optimise splitless time.

 
 

Analyte Focussing

The analyte is slowly introduced from the inlet during the whole splitless time (the inlet volume may be exchanged as few as two times during the 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 on the exact analytical conditions.  This would entirely negate any efficiency gained through the use of capillary columns and the resulting chromatographic peaks would be unacceptable broad.


To overcome these problems focussing techniques are used which involve setting the initial oven temperature at a suitably low value ensuring that condensation and re-concentration takes place in the column.


Two discrete focussing (cold trapping and solvent effect) mechanisms can be identified (Figure 9).

 

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Figure 9: Splitless injection analyte focussing.

 
 

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).

Solvent effect

Low boiling (more highly volatile) components remain dissolved in the solvent which also condenses in the inner wall of the GC column at low initial oven temperatures.  The solvent slowly evaporates to give a thin concentrated band of analyte (Figure 10).

 

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

 

Figure 10: Injection of n-alkanes in CS2 (B.P. 46 oC).  Increase in efficiency of early eluting (more volatile) analytes due to the solvent effect.

 
 

Solvent Choice

The solvent effect relies on the formation of a single contiguous film coating the inside wall of the capillary column.  This will only occur if the solvent polarity is matched to that of the stationary phase.  If this is not the case (e.g. using methanol as the sample solvent with a methyl silicone phase) the solvent will not condense as a film, but rather as droplets of solvent, each acting with an individual solvent effect.  This will lead to broad, split, or fronting and tailing peaks (the latter being known as the ‘Christmas Tree’ effect due to the triangular peak appearance).  These issues are generally most prevalent for earlier eluting (more volatile) analytes as later eluting analytes tend to be focussed via the cold trapping mechanism.  Solvent droplet formation usually only occurs when a critical solvent injection volume is exceeded (usually between 1 and 2 mL).  If a mismatch between the sample solvent and the column stationary phase is required (due to sample solubility characteristics) then a retention gap may be used.  This is a short piece of capillary column (0.5 – 3 m) which is coated using a phase which matches the sample solvent polarity.

As can be seen in Figure 11 the chromatogram exhibits poor peak shapes with a greater number of peaks than there are components in the sample.  Typically split peaks of this form are seen under the following circumstances

  • Poor column end cut.
  • Poor installation of the column into the inlet (typically the column is too high in the inlet).
  • Issues with peak focussing with splitless injection.
  • Damaged/occluded phase at the column inlet
  • Ferrule/septum shards stuck in the column inlet

Investigating the splitless conditions is a good place to start to troubleshoot a problem like that seen in Figure 11.  The initial oven temperature is good – 40 oC is more than 20 oC below the boiling point of methanol (64.7 oC).  The splitless time is a little long (2 mins.), however, it matches the initial isothermal hold in the method and while it could be a little shorter, which would sharpen the analyte peaks and reduce peak widths, it is probably not the cause of the gross peak deformation. 

However, the sample is dissolved in methanol which is very polar in comparison to the stationary phase (5% phenyl polydimethylsiloxane).  A simple change in solvent to dichloromethane (while keeping all other parameters the same) resulted in the chromatogram shown in Figure 12.

 

Figure 11: Poor peak shapes (split / shouldered) from the analysis of a test compound mix of analytes with various polarities in methanol using splitless injection.  Column: 5% Phenyl PDMS, 30 m x 0.25 mm x 0.25 μm.  Temp: 40 oC, 2 mins.  40 - 175 oC, 15 oC/min, 20 mins.  Inlet: Splitless (purge 2.0 mins.) 250 oC. 

Sample: 1 μL of 2 ppm each (1) 1-octanol, (2) n-undecane, (3) 2,6-dimethylphenol, (4) 2,6-dimethylaniline, (5) n-dodecane, (6) n-tridecane in methanol.

 
 

Figure 12: Chromatogram and conditions as per Figure 10 for the analytical test mix but sample solvent changed from methanol to dichloromethane.

 
 

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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 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 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 13).

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Figure 13: 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 the particular set of temperature and pressure conditions (Figure 14).

 

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Figure 14: Backflash calculator.

 
 

Table 3: Solvent properties.  Estimate volume expansion (VE) at 250 oC and 190 kPa.

 
 

The example splitless GC method (Table 2) can be used to demonstrate the use of a backflash calculator (Figure 15).  As can be seen from Figure 15 by inputting the parameters from the method the vapour volume can be estimated (36 μL),  this is well within the volume capacity of the liner used (800 μL), therefore, in this case there will be no risk of backflash.

 
Inlet liner   Deactivated double taper liner (V = 800 μL = 0.8 mL)
Injection volume   0.5 μL
Injection solvent   Acetonitrile
Head pressure   3.8 psi
Carrier   He, Constant flow 1.7 mL/min

Table 2: Example splitless GC method.

 
 

Figure 15: Agilent vapour volume calculator

http://www.chem.agilent.com/en-US/Technical-Support/Instruments-Systems/Gas-Chromatography/utilities/Pages/GCCalculators.aspx

 
 

Pressure Pulsed Injection

A technique known as pressure puled injection may be used to minimise 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 16).

 

Figure 16: Example of a pressure pulsed injection.

 
 

Analytical Chemists

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  • I can troubleshoot effectively
  • I know where to go for help
  • I understand my analyses
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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 volatilisation of all sample components.  This will ensure that temperature (inlet) discrimination of higher boiling (less volatile) analytes is minimised. 
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 oC.  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 analysed or where the sample is high boiling).

With the inlet set to 250 oC a scouting temperature gradient can be employed which will elute analytes and sample components over  a wide range of boiling points (Figure 17).  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 oC above this temperature to ensure efficient volatilisation.

 
 

Figure 17: GC temperature scouting gradient.

 
 

There are a number of chromatographic symptoms that may indicate problems with analyte thermal degradation, mostly involving a plateau at the front or rear of the chromatographic peak (Figure 18).  If this symptom is seen the inlet temperature may be reduced in 20 oC steps until the problem is resolved.

 
Figure 18: Typical peak shape of analyte inlet thermal degradation.
 

Optimisation of inlet temperature can be carried out by decreasing the inlet temperature in 20 oC 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 although it can never be truly ruled out.

 
 

Injector Temperature Profile

The metal body of the inlet is usually surrounded by a radiative heating material into which a heating element is placed.  It is important to establish a homogeneous heating profile over the whole length of the injector body to ensure that cold spots are not created at which less volatile analytes or sample components may condense and foul the inlet liner or other components.  In practice most inlets will have a temperature variation along the liner, however, for practical purposes this does not affect injection performance (Figure 19).

 

Figure 19: Typical injector temperature profile.

 
 

Inlet Discrimination

The phenomenon of sample discrimination leads to a non-representative sample entering the analytical column compared to the original sample (Figure 20). 

 

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Figure 20: Inlet discrimination.

 
 

Sample discrimination is shown in Figure 21, which illustrates the detector response to an injection of n-alkanes at equal concentration.  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 in 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, 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 21: Discrimination due to differences in boiling point.  Hot split injection of a solution containing equal amounts of normal alkanes in hexane.

 
 

Analytical Chemists

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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 22 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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Figure 22: GC liners.

 
 

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 23) are designed in such a way that sample vapourisation 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.

 

Figure 23: 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 vapourisation (Figure 24).

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

 

Figure 24: 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.  A good liner will:

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

The addition of quartz wool will increase the vapourisation 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 fibres are broken or when it becomes 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 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 25).  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 25: 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 vapourisation of high molecular weight compounds, and help to increase mixing of the sample vapour with the carrier gas.

 
 

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Cutting and Cleaning the Column

The column nut and ferrule should be placed onto the column prior to cutting at the inlet or detector end (Figure 26).  Failure to do so may risk shards of ferrule material entering the column and giving rise to poor peak shape.  Some operators prefer to use a septum to hold the column nut and ferrule in place (correct distance from the column end) during installation.  This should also be done prior to column cutting.

 

Figure 26: GC column configuration.

 
 

Using a scribe (ceramic wafer, diamond tipped pen), score the polyimide coating of the column and holding firmly just below the score, flick the column above the score away from you.  The wafer has two edges; the rough edge should be used to score the polyimide coating.  Usually 2.0 cm of severed column are enough to guarantee high quality stationary phase column although some workers prefer to take > 20 cm (Figure 27).
Fused silica column cutters can also be used for column cutting (Figure 27).

  1. Insert the capillary column (A) into the column cutter as indicated
  2. Fix the capillary column by turning the securing disk (B)
  3. Cut the column by turning the cutting disk (C), one full rotation is usually sufficient
  4. Use the magnifier (D) to verify the cut

Wipe the column with acetone to remove finger grease and other residues from the exterior surface of the column.

 

Figure 27: Column cutting.

 
 

Inspect the cut edge of the column with a 10 – 20x magnifier.  The cut end should be at a 90° angle relative to the column wall.  There should be no burs or large jagged areas (Figure 28).  If necessary, re-cut the column until a proper cut is obtained.  If a good column cut is not made then significant activity (resulting in poor quantitative precision) and split or tailing peaks may be observed in the chromatogram.

 

Figure 28: Good and bad column cuts.

 
 

Figure 29: Poor chromatographic peak shapes caused by poorly cut columns.

 
 

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Figure 30: GC column installation.

 
 

System Checks

Before installing the capillary column ensure old septa and injection port liners have been replaced with new ones.  Use cloves and/or clean tweezers to handle items to ensure cleanliness and inactivity of septa and liners.  Inspect gas purifiers for any colour change and change if required.

Place the Column in the GC Oven

Make sure the column tubing does not touch the sides of the oven.  If the column touches the hot oven wall the polyimide will ‘bake out’ and become brittle.  This may result in column breakage when the column cooling fan starts.  Unwind enough column at both sides to perform the installation.  In practical terms 30 cm (12 inches) of column at each end is usually sufficient to achieve proper column installation.

Making the Connection

Start by installing the column into the GC injection port.  Push the column through a septum keeping the septum at 90° to the column – this will be used to support the nut and ferrule at the correct point on the column whilst securing in the injector and detector.  Feed the column through the nut and ferrule prior to cutting the column. 
The use of a septum is not mandatory, however, it helps with the installation process.

Column Insertion Depth

The distances between the ferrule tip and the column inlet are crucial and differ between manufacturers.  Ensure that guidelines for column insertion distances are followed closely.  Use the septum to keep the nut and ferrule at the correct position in the column whilst inserting the column.  Note:  the GC instrument manufacturer MUST be consulted to find out what the correct column connection length is (either L1 or L2, Figure 31). 

 
 

Figure 31: Column insertion depth.

 
 

If the column is incorrectly positioned broad peaks or incorrect peak area ratios will be seen due to the increase in dead volume or incorrect sampling of the analytes into the column or analyte degradation (Figure 32).

 

Figure 32: Chromatograms produced from a capillary GC column installed at the wrong insertion depth.

 
 

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Over time the inlet end of a capillary GC column can become contaminated from the accumulation of non-volatile sample matrix components.  The phase in the front portion of the column can also be damaged from the continuous condensation and vapourisation of solvent and analytes.  Inevitably active analytes will adsorb to this contaminated/damaged section causing peak tailing (through secondary interactions), loss of resolution, loss of efficiency, and reduced analyte response.  When the chromatographic system degrades to an unacceptable level performance can often be restored by trimming the contaminated/damaged section from the inlet end of the column; typically at least 20 cm is removed from the column.  A very slight decrease in retention times and resolution occurs each time the column is trimmed due to the loss of theoretical plates.  Eventually the column will need to be replaced.

The use of a guard column is an inexpensive technique to extend the lifetime of capillary columns.  Typically a 1 - 10 m length of deactivated fused silica tubing attached to the inlet end of the column is used.  Deactivated fused silica tubing does not contain any stationary phase, however, the surface is deactivated to minimise solute interactions.  A suitable union is used to attach the tubing to the column.  If the tubing sizes are different it is better to have a larger diameter guard column or retention gap than a smaller one.  The onset of peak shape problems is the usual indicator that the guard column needs trimming or changing.

Retention gaps are used to improve peak shapes for some types of samples, columns, and GC conditions.  Usually a minimum of 3 – 5m of tubing is required to obtain the benefits of a retention gap.  The situations that benefit most from retention gaps are large volume injections (> 2 μL) and solvent-stationary phase mismatches for splitless, megabore direct, and on-column injections.  Peak shapes are sometimes distorted when using combinations of these conditions.  Polarity mismatches occur when the sample solvent and column stationary phase are very different in polarity.  The greatest improvement is seen for the peaks eluting closest to the solvent front or solutes with very similar polarity to the solvent.  The benefits of a retention gap are often unintentionally obtained when using a guard column.

 
 
 

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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 33).

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 oC (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
    • If the oven temperature is < 30 oC less than solvent B.P. use a hold time 
    • Start with 30 sec. and increase if necessary

     

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

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Figure 33: Setting initial oven temperature and hold time.

 
 

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Splitless injection
  • Split ratio should be optimized for each separation
  • A split of 100:1 is a good starting point
  • Always assess peak shape, column loading, sensitivity
Split injection
  • Optimize splitless time
  • For analyte focussing match the solvent polarity to the stationary phase polarity
Optimize injection volume to avoid backflash

Liners
  • Select correct liner for the injection mode
  • Monitor for liner deactivation
 
 
Column installation
  • Cut the column correctly (90o to the column wall)
  • Know the insertion depth for your instrument
  • Use a guard column to extend column lifetime
Optimize initial oven temperature and hold time
  • Split injection does not normally require an initial hold time
  • Hold time for splitless injection should match the splitless time
  • Lowering the initial oven temperature is better than adding a hold time
 
 

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Learn chromatography from the experts

Whether you work in a lab or manage a lab, you will benefit from being a member of CHROMacademy.

As a member of CHROMacademy, you will get access to our vast e-Learning archive full of great interactive content and animations.
All our Essential Guide Webcasts and tutorials and LCGCs archive of magazine articles and webcasts from your favourite authors - John Dolan, John Hinshaw, Mike Balough, and Ron Majors. Plus vendor application notes, electronic laboratory tools and calculators and with our 'Ask the Expert' function - help is always at hand.

 

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The Essential Guide from LCGC’s CHROMacademy presents the first in a series of webcasts aimed to develop the skills and understanding of GC users. In this session, Dr. John Hinshaw (Senior Scientist, BPL Global Ltd) and Tony Taylor (Technical Director, Crawford Scientific) take an in-depth look at principles of GC sample introduction and the associated variables which need to be defined in GC methods. We describe the principles of sample injection and describe in detail how method variables are set and optimized using real life ‘good’ and ‘bad’ method examples.

Tony Taylor
Technical Director
Crawford Scientific

John V Hinshaw
Senior Scientist
BPL Global Ltd

Key Learning Objectives:

  • Understand the working principles of the Split / Splitless Injector
  • Appreciate the major and minor variables which control sample injection
  • Recognize when a method is deficient in terms of the amount of information given and make informed choices about the parameters which are not stipulated
  • Appreciate the importance of proper inlet function and make informed choices in terms of inlet liners
  • Understand how to set and optimize sample injection variables based on sample (analyte) type and intended analytical outcomes