No thanks! I would like to know more about CHROMacademy

 Over 3000 E-Learning topics / 5000 Articles & Applications
 
How to Optimize Key Variables in GC Analysis - Sample Introduction

Every analysis can benefit from the best injection possible. This involves selecting the ideal injection technique and optimizing it for every sample.

This article will cover which injection technique can be used for different sample and analysis types, which parameters should be optimized, and also some of the drawbacks of the inlet.

Split/Splitless Injection

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 by performing an 'on-instrument' dilution (Figure 1).

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 1: Split injection.


However, why perform an on-instrument dilution rather than just diluting the sample further prior to injection?

Capillary GC columns are limited to the amount of each analyte which can be introduced onto the column before peak shapes begin to deteriorate.  Smaller i.d. columns and thinner stationary phase films have lower capacity, and analyte concentrations in the order of a few nanograms on column are typical, therefore, we require a reasonably dilute sample (Table 1).

 
Column i.d. (mm) Film Thickness (µm)
0.1 0.25 0.5 1.0
0.10 10 ng 30-40 ng 50-70 ng 100-200 ng
0.18 20-30 60-80 100-150 250-350
0.25 30-40 125-175 175-250 400-500
0.32 50-70 200-250 250-350 600-800
0.45 80-100 300-400 400-500 800-1000
0.53 100-120 400-500 500-700 1000-15000

Table 1: Typical column capacities for capillary GC columns. Mass (ng) per analyte.

Performing an on-instrument dilution is preferable as it results in sharper peaks.  The split ratio is used to control the amount of analyte reaching the column, which ultimately affects peak width and sensitivity (Figure 2).

Typical split ratios are in the range 1:20 to 1:400.  When using thick stationary phase film (>0.5 μm) or wide bore (0.533 mm i.d.) columns the sample capacity increases and lower split ratios are typical, 1:5 to 1:20.  With very narrow GC columns (<100 μm i.d.) split ratios can be as high as 1:1000+.

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player


Figure 2: Setting the split ratio.


Splitless injection is used for trace analysis as the entire sample is transferred to the analytical column (Figure 3). 

 

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 3: Splitless injection.


Analyte transfer from the inlet to the column is slow which would result in broad analyte peaks, however, two focusing mechanisms occur which mitigate this problem and are a must for optimizing this injection technique (Figure 4).

These involve setting the initial oven temperature 20 °C below the sample solvent boiling point which ensures that condensation and re-concentration of the analyte band takes place in the column.  The sample solvent and column polarity should also be matched, for example, non-polar hexane as the solvent when using a non-polar PDMS column.

 

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 4: Splitless injection focusing mechanisms.


The splitless time should be optimized; too short a splitless time and high boiling analytes will be lost, too long a splitless time risks a large solvent peak (Figure 5). 

Typical splitless times are in the region 20-90 seconds.

 

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 5: Optimizing splitless time.


Sample discrimination (Figure 6) and sample degradation occur under both split and splitless injection modes. 

Due to the vaporizing nature of the inlet solvent backflash (Figure 7) can occur causing sample loss, poor resolution, peak shape problems, carry over, and ghost peaks if the injection volume is not optimized based on the particular sample solvent and inlet conditions (temperature, pressure, and liner volume).

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 6: Sample discrimination.

 

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 7: Solvent backflash.


 

Cool-on-Column

Cool-on-column injection is particularly suited to mixtures containing high and low volatility analytes and for trace analysis where quantitative reproducibility is important.  The sample solvent is deposited directly on to the column giving high sensitivity (Figure 8).

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 8: Cool-on-column inlet.


The initial oven and inlet temperature should be set 10-20 °C below the sample solvent boiling point, to allow focusing of the analyte band, which also has the added advantage of reducing sample discrimination and thermal degradation. 

Matching column and sample solvent polarity is also essential to allow a homogeneous solvent film to be formed in the column; if the polarities are different broad and often split peaks will be observed throughout the entire chromatogram (Figure 9).

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 9: Chromatographic peak deterioration due to mismatch of injection solvent and capillary column polarity.


As the entire sample is introduced on to the column the use of a retention gap serves to protect the analytical column from involatile sample components, whilst also allowing increased injection volumes (2-5 μL) by providing a large surface area for solvent film formation (Figure 10).

Regardless of column internal diameter a wide bore (0.53 mm) retention gap must be used.  This technique can be prone to sample overload, is difficult to use with columns less than 0.25 mm i.d., and is complex to automate.

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 10: Use of a retention gap for cool-on-column injection.


Programmed Thermal Vaporizing

This inlet is best suited to the injection of large sample volumes (100 μL is possible, with injections of up to 1 mL having been demonstrated).  In solvent vent mode, when carrying out multiple small injections, the time interval should be sufficiently long to allow almost the entire sample solvent to evaporate.  The optimum interval time is generally in the range 2-20 s.

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 11: Programmed thermal vaporizing inlet.

 

For injection volumes below 10 μL use an unpacked baffled liner.  For larger volumes use glass wool or beads packed in a straight liner so that the needle just touches the packing to help both solvent evaporation and analyte trapping.  Liners with selective adsorption materials (Tenax TA™ or Carbotrap™) increase the range of components which can be trapped (improved trapping of volatiles). 

The best results are obtained when the boiling point difference between the sample solvent and analytes is at least 150 °C, with lower boiling solvents (<120 °C) being optimum.  The initial inlet temperature should be set 30 °C below the solvent boiling point.  Normal split ratios are employed (50:1 to 200:1).

In general lower temperatures with higher flows are more desirable.  Due to the number of parameters which must be optimized this is the most complex injection technique, which also makes it expensive.  If the parameters are not correctly optimized there can be a loss of volatiles when using solvent vent mode.


You may also enjoy the following CHROMacademy content

eLearning
Sample Introduction

Webcasts & Tutorials
What GC Operators Need to Know: From Gas Supply and Inlets to Column Selection and Temperature Programming
Everything You Should Know About Headspace GC
GC Troubleshooting - Gas Supply and Inlet Issues
Split/Splitless Injection

Quick Guides
5 Ways to Improve Your Split/Splitless Injection Technique
GC Setup Checklist
The Why, What, and How of Headspace GC
Best Practices for GC Sample Introduction


loading data
loading data
loading data
loading data
loading data


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.

group  subsCHROMacademy can deliver to corporate clients on a multi-user subscription basis.
Served up from secure servers to the corporate intranet or individual desktops.

  • Microsite - your own learning site powered by CHROMacademy
  • Your Landing Pages -with your logo and branding
  • Customized Assessments - Based on content agreed upon Certificate of Completion
  • Certification Programs - Offer your learners a goal to strive towards
  • Our Learning Management System is S.C.O.R.M. compliant and will connect to your system
  • Engagement Package - E-newsletter stimulation program derived from your content and ours
  • Full archive of Essential Guide webcasts & tutorials
  • 1000’s of eLearning topics - HPLC / GC / Sample Prep / Mass Spec
  • Ask the Expert - our experts will answer your chromatography questions within 24 hrs.
  • Assessments - test your knowledge
  • Application notes & LCGC articles
  • Troubleshooting and virtual lab tools

Request a quote

 

 
 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

loading data

loading data

loading data

 

loading data


loading data