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5 More Pictures Which Reveal Problems With Your GC Analysis – And How to Fix Them!

A picture paints a thousand words.  The art of GC troubleshooting often lies in being able to recognize a problem from the evidence presented in the chromatogram or baseline appearance.  In this short series of two articles, we present 10 simple GC pictures which show you how to recognize the issues, deal with the causes, and prevent them from happening again.  Here we present another five pictures.

1) Irreproducible Retention Times

Irreproducible Retention Times

There are two types of retention time variability that can be observed.  Firstly, from injection to injection, this could be from injection to injection in a run of samples or multiple injections from the same vial.  This may be caused by changes in carrier gas linear velocity due to a defective electronic flow controller, leaking septum or column connection, or variable gas supply due to a leak in the gas supply tubing.  Under these circumstances you would expect to see variable retention times for all analytes. 
To remedy the problem ensure that the instrument settings are correct and occasionally verify that the instrument calculated flow/linear velocity matches the carrier gas flow by measuring all inlet flows using a flow meter.

Other causes of this type of variable retention time are variations in the oven temperature (either from a faulty temperature controller or from insufficient thermal equilibration time) or if the inlet pressure is not high enough to sustain flow at high gradient temperatures.  In constant flow operating mode, the carrier pressure is ramped to attain a constant flow through the temperature program (as the temperature is increased carrier gas viscosity also increases, and therefore, requires a higher pressure to maintain constant flow), in constant pressure mode the inlet pressure remains constant which means that as the carrier gas viscosity increases during the temperature program the linear velocity will decrease causing retention times to change.  With mass-flow or flow sensitive detectors, this phenomenon can also manifest itself as either a gradually increasing or decreasing baseline signal depending upon the response characteristics of the detector.

Therefore, it is often better to use constant flow mode to avoid these problems.  Figure 1 demonstrates the improvement in chromatography when using constant flow mode; there has been an overall gain in sensitivity (peak areas have increased) mainly due to the decrease in peak width, the rising baseline has been eliminated due to the constant flow into the mass-flow sensitive detector which allows for more reproducible integration and quantitation, and finally retention time has decreases by a factor of three.

Constant flow vs. constant pressure analysis

Figure 1: Constant flow vs. constant pressure analysis.

If you have long term variability in retention times, this may be attributed to changes in the column, such as, stationary phase degradation or shortened column due to trimming which when entered incorrectly into the GC data system causes the instrument to incorrectly calculate the linear velocity of the carrier gas.  Remember, the EPC calculates all flows from the applied pressure, the nature of the carrier gas, and the column dimensions.

However, how important is it to know the exact length of the column?  Columns are regularly cut by users, with varying unspecified lengths being removed. It would be impractical to expect users to record the exact length of the small pieces of column that are generally removed and it is totally impractical to measure the length of the existing column - the standard length being 30 m. Having to unwind and rewind 30 m would likely damage the column.  In general removal of these small portions has negligible effect on the performance of the column (e.g. removal of a typical length of 3 cm from a 30 m column equates to 0.1% of total length). 

The assessment of exact column length is of no benefit whatsoever for the majority of applications and is better served by applying meaningful system suitability criteria to critical parameters within the method that are affected by reducing the column length - this would be absolute retention time, resolution, and possibly some effect on peak area due to slight changes in the split ratio. The criticality of these parameters has to be assessed on a method by method basis and adequate provisions made for system suitability criteria that are relevant, meaningful, and have sensible limits applied. In general these would be tests such as retention time limits, resolution limits, and linearity.

There are certain cases where reduction in column length is more significant - either with short columns (10 m or less) or columns which are exposed to excessive contamination and need larger lengths removed more regularly. This should be captured by system suitability or by use of retention gaps.  If the column is too short, retention times will be too short and vice versa.


2) Integration Issues

Integration Issues

In this case it is difficult to reliably integrate the peaks as there is a rising baseline.  The first solution to avoid this problem would be to eliminate any type of baseline drift in the first place.  This can be done by reducing column bleed by properly conditioning the column, using constant flow or linear velocity mode especially when using a temperature program, or if you need to use constant pressure mode and are using a mass-flow sensitive ionizing detector you may have the ability to use variable make-up flow to compensate for the change in linear velocity during the method. 

A great tip to help condition the column correctly and in the most efficient manner is, once the column has been connected to the inlet do not connect it to the detector and allow at least six column volumes of carrier gas to pass through the column in order to remove air from the column as well as sparging any dissolved oxygen from within the stationary phase (Table 1).  This will allow you to condition the column for less time and at a lower temperature.  Once this step has been completed, ramp the oven temperature (ensure the carrier gas is still flowing through the column) at 20 °C/min. to 20 °C above the upper temperature required by the analytical method.  Once the upper temperature limit has been reached the column should be conditioned for the correct amount of time based on the dimensions and phase type (Table 2).

Column Internal Diameter (mm) Minimum Flow Rate (mL/min.) Minimum Purge Time (min.)
0.53 5 10
0.32 1.5 20
0.25 1 25
0.18 0.8 30
0.1 0.5 40

Table 1: GC column purge times.

 
Phase Type Length (m) Film Thickness (mm) Conditioning Time (min.)
Non-polar < 30 <0.5
0.5-1.0
>1.0
15
30
60
30-60 <0.5
0.5-1.0
>1.0
30
45
60
>60 m <0.5
0.5-1.0
>1.0
60
90
120
Mid-polarity < 30 <0.5
0.5-1.0
>1.0
20
40
60
30-60 <0.5
0.5-1.0
>1.0
40
60
80
>60 m <0.5
0.5-1.0
>1.0
80
120
160
Polar < 30 <0.5
0.5-1.0
>1.0
30
45
60
30-60 <0.5
0.5-1.0
>1.0
60
90
120
>60 m <0.5
0.5-1.0
>1.0
80
120
160

Table 2: Recommended conditioning times for various capillary GC column types.


It is always good policy to record the bleed profile for a column when new (Figure 2), so that the level of bleed can be referenced at a later date in order to assess the degradation of the stationary phase over time, and perhaps a performance limit established for column replacement.  Simply run the method without making an injection or inject a small amount of sample solvent.  If you are using mass spectral detectors, the presence of ions at m/z 207, 281, and 355 indicate column bleed.  These will almost always be there – it is whether they are causing problems either spectrally or in terms of reproducibility of integration that really matters.

capillary GC stationary phase bleed

Figure 2: Typical capillary GC stationary phase bleed profile for two different polysiloxane based stationary phases.


Another approach to avoiding column bleed is to consider the stationary phase and column type you are using.  High polarity columns inherently bleed more; therefore, using the lowest polarity column possible can help to avoid some unwanted bleed.  You can also consider using a GC-MS designated column (even if you are running a GC method) as they have been designed to be low bleed.  They will provide better sensitivity due to the improved signal to noise ratio and will give better mass spectral purity from the absence of background bleed ions (Figure 3).

GC and GC-MS designated column
Figure 3: Comparison of 35% phenyl standard GC and GC-MS designated column.

In the real world rising baselines are often inevitable, therefore, spending some time familiarizing yourself with the advanced integration settings in your data system will allow you to properly integrate challenging chromatograms - these settings include threshold, slope sensitivity, baseline reset points, and different integration methods, such as, valley-to-valley.  Remember to always use the same integration method every time to provide consistent results (Figure 4 and 5).

Common peak integration errors

Figure 4: Common peak integration errors.

Peak integration methods

Figure 5: Peak integration methods. 1) Drop perpendicular, 2) valley to valley, 3) tangential skim, 4) exponential skim, 5) Gaussian skim


3) Step-Shaped Peaks

Step-Shaped Peaks

The step-shape can either be before or after the peak, and may also be accompanied by tailing or a shoulder, and is due to analyte thermal degradation in the inlet.  To remedy this problem reduce the inlet temperature in 20 °C steps until a normal peak shape is achieved.  However, take care not to lower the inlet to a temperature that is too low to achieve proper volatilization of all of your analytes, if this happens you may begin to see irreproducible peak areas.
To choose an appropriate inlet temperature the following steps can be followed:

  • A good starting point for method development and for new analyte applications is 250 °C
  • A scouting temperature program can be used to estimate the elution temperature of the highest boiling component (Figure 6). 
    Set the inlet temperature at least 50 °C above this temperature to ensure sufficient sample volatilization
Scouting temperature program

Figure 6: Scouting temperature program.


 4) Poor Peak Shape (Early Eluting Peaks)

Early Eluting Peaks

Poor peak shape for early eluting analytes when you are carrying out a splitless injection is caused by a sample solvent/column polarity mismatch or the wrong initial oven temperature (the oven temperature is too high).  In splitless injection the sample transfer from the liner to the column is slow; therefore, to mitigate band broadening effects from this the sample must be refocused at the head of the column.  Two focusing mechanisms occur (Figure 7):

  1. Solvent focusing, where low boiling analytes remain dissolved in the solvent which condenses on the inner wall of the GC column at low initial oven temperatures.  The solvent polarity must match that of the column to ensure a contiguous film is deposited leaving a narrow sample band when the solvent evaporates

  2. Cold trapping, where higher boiling analytes are condensed in a tight band in the temperature gradient between the inlet and the column oven.  Initial oven temperatures should be set 20 °C lower than the boiling point of the solvent used to dissolve the sample

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Figure 7: Splitless injection focusing mechanisms.

5) Loss of Resolution From Loss of Efficiency

Loss of Resolution

In order to diagnose this problem carry out a plate count (your software will probably do this) on the peaks.  If you are seeing a loss in efficiency the plate count will reduce, in particular for early eluting peaks (Figure 8). 

There are several causes of this problem including, poor column cut and installation which leads to analytes being held up at the entrance to the column resulting in a broadened peak, loss or contamination of the stationary phase (this can be resolved by trimming the column), or the incorrect column length or diameter entered into the data system which results in an incorrect carrier gas velocity which in turn affects efficiency (remember each carrier gas has an optimum linear velocity to produce chromatography with the best efficiency).

Plate count
Figure 8: Plate count.

Where:
tr = retention time
Wb = peak width at base
W1/2 = peak width at half height

Internal Diameter (mm) Film Thickness (μm) Column Length (m) Theoretical Plates Theoretical Plates per Meter N/m*
0.18 0.18 20 133200 6660
0.25 0.25 30 138900 4630
0.32 0.32 30 112800 3760

Table 3: Typical column efficiencies. *Measured with a k = 5.


There may be other causes of these problems, so for further troubleshooting tips visit the CHROMacademy GC Troubleshooter »

Watch the associated webcast: GC Troubleshooting in 20 Pictures »

You may also like...

Part 1 of this series

5 Simple Pictures Which Reveal Problems With Your GC Analysis – And How to Fix Them! »

Webcasts & Tutorials

GC Troubleshooting in 20 Pictures - Part 1 »

GC Troubleshooting Masterclass »

Quick Guides

5 Ways to Improve Your Split/Splitless Injection Technique »

GC Column Installation and Conditioning Guide »

Chromatography Troubleshooting Tips »

GC Column Maintenance - Prevention is Better than Cure »

10 Common GC Mistakes »

Troubleshooting Videos

Practical GC Troubleshooting Video - Peak Splitting »

Practical GC Troubleshooting Video - Peak Tailing »

Practical GC Troubleshooting Video - Peak Area Reproducibility »

GC Troubleshooting Video Guide to Irreproducible Retention Times »

 
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Dr. Dawn Watson
 

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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