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GC Troubleshooting Guide to
Peak Area Reproducibility and Reduction of Sensitivity

The Problem

By just looking at the chromatogram from this problem there doesn’t seem to be an issue, we have good peak shapes and consistent retention times for our analytes (Figure 1).  However, the quantitation is a disaster.  The peak areas are highly irreproducible for the higher boiling, later eluting analytes which also show a decrease in sensitivity (Table 1).

Column: 5% Phenyl PDMS.  10 m x 0.53 mm x 2.65 µm
Temperature: 70-77 °C, 1 °C/min., 0 min.
77-225 °C, 10 °C/min., 22 min.
225-275 °C, 20 °C/min., 10 min.
Carrier: He at 10.5 mL/min.
Inlet: Splitless (purge 60 mL/min. at 1.0 min.)
Splitless 4 mm liner
300 °C
Detector: FID
H2, 30 mL/min.
Air, 300 mL/min.
He, 21 mL/min.
 

This page is part of our series of 6 free Chromatography Troubleshooting Guides.

HPLC

Changes in Selectivity, Retention
and Resolution »

Cycling Baselines and Pressure Fluctuations »

Solving HPLC Peak Splitting Problems »

GC

Broad Peaks and Loss of MS Sensitivity »

Broad, Double/Split, Fronting Peaks »

Peak Area Reproducibility and Reduction
of Sensitivity »

Figure 1: Separation of a number of long chain alkanes and alcohols using the conditions shown below, in which peak area reproducibility of higher boiling analytes is problematic.

 

 

Benzyl alcohol

Phenylethanol

Docosanol

Octacosane

 

377598.41

358139.35

629958.09

652108.72

 

365602.43

346592.41

588086.01

606098.41

 

368952.65

349396.18

627075.09

652670.23

 

375923.95

355689.95

632828.19

654923.09

 

366439.31

347266.76

591502.48

614188.56

 

368243.53

349041.6

566519.12

573894.65

Mean

370460.0467

351021.0417

605994.83

625647.2767

SD

5054.867448

4748.363772

27668.50239

33103.82449

% RSD

1.364483834

1.352729098

4.565798423

5.291132196

Table 1: Peak area data for each analyte over several injections, mean, standard deviation (SD), and relative standard deviation (%RSD).  Relative standard deviation of peak areas of higher boiling analytes is unacceptably high.

 

The CHROMacademy GC Troubleshooter

The symptoms within the troubleshooter are grouped by type (i.e. baseline issues, peak shape, issues, and retention issues) with each symptom being accompanied by an illustration to provide clarity. Our problem is with quantitation, hence, the symptoms can be found under the separation and quantitation issues group.

We are able to select up to three chromatographic symptoms at once which is one of the unique troubleshooter features, in order to reach a more accurate diagnosis of the problem. In this case we selected ‘sensitivity reduction - later eluting peaks’ and ‘peak area reproducibility poor’ (Figure 2).

Figure 2: Input of both chromatographic symptoms into the CHROMacademy GC Troubleshooter.

 

With very little work we now have a list of possible causes and more importantly possible solutions to our problem (Figure 3). Problems can often have multiple causes and when you combine symptoms the number of causes increases; it is this which often makes troubleshooting seem like a daunting task.

However, the possible causes reported by the troubleshooter algorithm are ordered depending on how many of the symptoms are matched and the likelihood of that group of symptoms giving rise to certain problems. This unique, proprietary rating system is very important as it helps to narrow down the number of possible causes.

 

Figure 3: CHROMacademy GC Troubleshooter results page, including causes of poor peak area reproducibility and sensitivity reduction,
solutions to each individual cause, and further resources for resolving the problem.

 

The Solution

Luckily this turned out to be a fairly straightforward problem.  Often when poor reproducibility is seen with higher boiling analytes, inlet discrimination is the cause.  Discrimination describes the phemomenon in which only a portion of certain analytes are transferred to the column from the inlet, typically as with our chromatogram, the response is lower for high boiling analytes.  Discrimination can be caused by a number of factors including:

  • Slow autosampler injection, causing adsorption of higher boiling analytes onto the inside or outside surface of the injection syringe.
  • Failure to ‘wipe’ the autosampler syringe with the liner packing.
  • Liner packing (glass wool) omitted or in the wrong position to act as a surface for condensation/evaporation for higher boiling analytes.
  • Splitless time too short (purge time too early) in splitless injection.

There are also several solutions to the problem:

  • Use a ‘fast’ autosampler setting to ensure the sample is ejected into the inlet in the liquid phase (no pre-volatilization inside the syringe needle).

OR

  • Increase the residence time in the inlet for the syringe to ensure that all high boiling components adsorbed onto the syringe surfaces have time to evaporate.
  • Introduce a plug of deactivated glass wool in the correct place into the liner to ‘wipe’ adsorbed components from the syringe tip.
  • Ensure the splitless time is optimized.

It should be noted in this case that the third cause in our list is the same as one of the sources of inlet discrimination ‘splitless (purge on) time too short.  Therefore, the first thing we tested was the effect of the splitless time.  This was carried out by extending the splitless time from 60 seconds to 90 seconds.  The following results were obtained (Table 2): 

 

Benzyl alcohol

Phenylethanol

Docosanol

Octacosane

 

317827

343742

844729

894098

 

312422

339093

820911

898504

 

315515

341143

831458

880334

 

319106

346673

851261

909819

 

319352

343603

846172

908329

 

317029

343042

834228

902576

Mean

316875.2

342882.7

838137.5

898943.3

SD

2597.0

2570.5

11266.0

10858.6

% RSD

0.8

0.7

1.3

1.2

 

Table 2 shows that the data obtained indicate much more acceptable precision. 
The end user may further investigate inlet temperature or a slightly longer splitless time (120 sec.) to obtain the best precision, however, one needs to guard against broad tailing solvent peaks and rising baselines throughout the run when using very long splitless times. 

To optimize the splitless time, it is usual to measure the peak area reproducibility of early, mid, and later eluting compounds whilst increasing the splitless time (purge on time) until the peak areas become consistently reproducible.  This should represent a good compromise between the inlet being emptied of all analyte components and having a reasonably shaped solvent peak and baseline.

For more information on optimizing splitless time see:

Theory and Instrumentation of GC / Sample Introduction / Splitless Injection »

 

Have you ever had a problem with the following symptoms?

Drifting baseline

 

 FID: Flame ignition problems

Use the CHROMacademy GC Troubleshooter to see what could be causing the problem and how to resolve it.

GC troubleshooting

 
 

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