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5 ways to improve your Split / Splitless Injection technique
1. Understand the process

 

 

Split/splitless injectors typically vaporize a sample dissolved in a suitable organic solvent under increased temperature.  The sample vapors are entrained into the carrier gas flow inside a 'liner' or 'sleeve' within the inlet and from there pass into the column or out of the inlet via the 'split' line/valve (Figure 1).

 

Figure 1: Typical split/splitless inlet. »

 

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Split injection is used to perform an 'on-instrument' dilution and the relative amounts of sample which enter the column or are discarded to waste via the split line are adjusted using the relative carrier and split flow rate ratio (Figure 2).  See point 3 for why we use split injection and don't just dilute the sample further prior to injection

 

Figure 2: Split/splitless inlet operating in split mode. »

 

 

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Splitless injection is used when analyte concentrations are low i.e. for trace analysis.  In splitless injection the split valve is initially closed, ensuring that all sample passes onto the column, then at an optimized time the split line is turned on to clear the inlet of any residual vapors (Figure 3).  The transfer of the sample vapor (diluted with carrier gas) from the inlet is much slower compared with split injection.  This can result in band broadening, therefore, the sample vapors must be trapped (condensed) at the head of the column by using a low initial oven temperature.

 

Figure 3: Split/splitless inlet operating in split mode. »

 

 

 

 

 

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2. Septa and Septum Purge

Septa are plasticized rubber disks, held under torque, which form a seal around the injection syringe needle so that pressure is maintained during sample introduction (Figure 4).  They need to be highly 'plastic' in order to do this effectively and, therefore, many have plasticizers added.  These components leach out of the septum and can give rise to noisy baselines and, under gradient temperature programming conditions, discrete interference peaks (Figure 5).  The septum purge gas flows over the underside of the septum to carry away these bleed products and results in better baselines, fewer 'unknown' peaks, and better detection limits. 

     

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Figure 4:  GC inlet septum.

 

Figure 5: Septa problems.

 

When was the last time you checked the septum purge flow settings or verified the flow via your instrument EPC system or even better with an electronic flow meter attached to the septum purge outlet?  You'd be amazed at how many systems have blocked septum purge lines which contribute to lower detection limits or noisy baselines and which go largely undiscovered!

Septa perish over time and become cored.  This can result in small shards being released into the inlet which can lead to the problems outlined above; however, the septum purge flow cannot mitigate problems caused by a cored septum.  In order to avoid these problems do not leave changing your septum until the system will not hold pressure – change regularly to avoid reactive maintenance and poor chromatography.  Further, most septa are held under torque using a nut arrangement, make sure to follow the manufacturers guidelines to the level of torque required – too much will mean shorter septum lifetime and too little will result in a leaky system.   Septa are rated to different upper temperature limits and some are faced with a thin layer of PTFE to restrict the outgassing of potential contaminants.

3. It's all in the split!

Why do we need split injection, why perform an on-instrument dilution rather than just diluting the sample further prior to injection?

Firstly, capillary GC columns are limited to the amount of each analyte which can be introduced onto the column before peak shapes begin to deteriorate.  This is known as sample loading capacity.  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 (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 1000-1500
« Table 1: Typical column capacities for capillary
GC columns. Mass (ng) is per analyte.
 

 

 

 

Therefore, we require a reasonably dilute sample.  Performing an on-instrument dilution is preferable as it results in sharper (more efficient) peaks.  The flow of carrier gas through the liner is higher in split injection, which carries the sample into the column more quickly (i.e. in a narrower band) and, therefore, results in less analyte (peak) dispersion in the inlet. The higher the split flow, the faster the analyte is carried onto the column, and hence, the sharper are the peaks.  Of course, method detection limits should be considered when increasing split flows as sample concentration on column will be reduced and the gain in sensitivity due to the increased peak signal to noise ratio may be outweighed by the reduction in absolute sample amount on-column.

In an experiment where 1 µl of solvent is introduced and expands to give 1 mL of sample vapor in the inlet, a split flow of 100 mL/min and a carrier flow of 1 mL/min will result in a split ratio of approximately 100:1 (Figure 6).  This would mean, in most common instrument designs, a flow through the liner (containing the sample) of 101 mL/min.  Our 1 mL of sample vapor would therefore be transported onto the column in 1/101 x 60 seconds - approximately 0.6 seconds (a very good initial peak width on column I'm sure you will agree).  When the split ratio is reduced to 10:1, the sample introduction would take 1/11 x 60 seconds – approximately 6 seconds which is not so good, but obviously still OK for most applications.

 

Figure 6: Setting the split ratio. »

 

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4. The golden rules of splitless injection

These rules are vital to getting the best detection limits and quantitative performance from your equipment.

Initial oven temperature setting - should be 20 °C lower than the boiling point of the solvent used to dissolve the sample (sample solvent).  In splitless injection the sample vapor moves very slowly into the column and we need to have a mechanism to re-focus the analyte band to avoid very low peak efficiencies.  Having a low initial oven temperature traps (focuses) the analyte as it enters the column – sharpening up the analyte band (chromatographic peak) and improving the peak shape and efficiency (Figure 7).

 

 

 

 

 

Match the sample solvent and column polarity - in order to avoid very broad
or even split / shouldered peaks (Figure 7).  Hexane with 100% PDMS (non-polar)
column or methanol with a Wax (polar) column for example.

 

Figure 7: Analyte focusing in splitless injection. »

 

 

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Optimize the splitless time – at some point after injection the split line valve MUST be opened to eject lingering components from the inlet which would otherwise lead to oversize and tailing solvent peaks and rising baselines throughout the analysis.  The splitless time should be determined by monitoring the peak area of analytes at the beginning middle and end of the chromatogram and plotting peak area versus splitless time.  Try values in 10 second increments from 10 seconds after injection (10, 20, 30 seconds etc.) and choose a value which shows  a repeatable peak area to the previous time point for the best compromise between chromatographic peak shape and area reproducibility (Figure 8).

 

Figure 8: Optimizing splitless time. »

 

 

 

 

 

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Avoid backflash – excess sample vapor will contaminate your system and cause carry-over and peak area irreproducibility.  Use a backflash calculator (Figure 9) to ensure that the volume of sample vapor you create is lower than the available volume within the inlet liner to avoid these issues (this is important in split and splitless injection).

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Figure 9: Backflash calculator.

5. It's a little wooly

Choice of liner type and packing material is important to ensure good injection reproducibility.  There are several aspects which need to be considered.

Glass wool packing – deactivated glass wool is used to provide a large surface area from which less volatile components may volatilize, it provides a tortuous route through the liner so that the sample vapor is properly mixed to ensure good homogeneity, and also serves to wipe the tip of the syringe to avoid the discrimination processes whereby higher boiling analytes are selectively removed from the inlet prior to them volatilizing.  For all these reasons, deactivated glass wool in the right amount, density, and positioning within the inlet liner can vastly improve the precision and accuracy of sample introduction, especially in splitless injection.  If you are unsure of the right amount or positioning of the glass wool please contact your manufacturer.

Liner features – a gooseneck restriction at the bottom of the liner prevents analytes encountering hot metal surfaces and potentially degrading, which is especially useful in splitless injection.  A gooseneck at the top of the liner prevents overfilling of the liner (backflash) and may improve analytical reproducibility.

For split injection, the default choice is a straight through (no gooseneck) unpacked liner.  For splitless injection the default choice is a packed liner with a lower gooseneck.

For more information on liner selection see the following CHROMacademy page: GC Inlet Systems »

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