Retention Time Issues in VUV Analyze Software Applications

In a recent post, we covered rising baselines and the problems they can cause when doing analysis with VUV Analyze™ Software.  In this post we will discuss another common problem affecting VUV Analyze results, which is when the analyte retention times in the run do not correspond to the times predicted by the retention index (RI) file.  We will discuss symptoms of RI file issues, how to go about confirming the problem, and steps to correct the issue.

RI Files and Time Interval Deconvolution™ (TID™)

VUV Analyze works using Time Interval Deconvolution (TID).  The chromatogram is broken down into regularly spaced time intervals, each with a unique summed absorbance spectrum. TID then determines what analyte(s) make up the spectrum in each time slice.  When the library search is done to match the spectrum of a time slice, the search is limited to the analytes expected to elute within a set time window of the time slice being examined. The RI file is how VUV Analyze knows what analytes to look for in the given time slice. If there is a discrepancy between when analytes are eluting and when they are expected to elute based on the RI file, the analysis will not be accurate.  This can appear as a high area reject percentage or as inaccurate results.

Examining a run file for poorly fit time slices

To see exactly how a retention time issue could affect analysis, we will want to have a closer look at the individual time slices analyzed.  After  VUV Analyze has analyzed a run from production mode, we will change to R&D Mode, accessed by pressing Control+Shift+R. When reviewing time slices it is helpful view the Log Chi² values as well as the fit statuses of the time slices in the chromatogram plot.  To add these features, we first click the gear icon, bringing up the Graph Configuration window, and make the Log Chi² values visible in the Error Metrics tab (Figure 1). Next, we left click on the top and bottom values on the Y-axis to set those to range from -9 at the bottom of the plot to 0 at the top.  Then we add all fit statuses and commit the changes (Figure 2).

Figure 1. Adding error metrics to chromatogram plot.

Figure 2. Adding Fit Statuses to chromatogram plot.

Poor fits are noticeable either by a high Log Chi² value, when the fit is not so bad that a time slice is rejected, or by a red fit status, when the time slice did not meet the minimum fit required by the analysis parameters.  Log Chi² values generally rise and fall with the peak intensity.  When we see either a red fit status or high Log Chi² at either end of a peak, this can indicate that the analyte in the peak was not fully covered by the retention index window of the search.  For example, in Figure 3 we see that the time slice at ~2.61 min has a higher Log Chi² value than the time slices around it.

Figure 3. Unexpectedly high Log Chi2 value for time slice at the beginning of a peak

We next select the cursor tool and then click in the chromatogram plot on this time slice to view it.  Going to the spectra tab  (Figure 4), we see that the fit area (blue fill) does not match with the absorbance spectrum of the time slice (green line). Clicking on the adjacent time slice to the right, we can see that the analysis did a good job of fitting a spectrum with that same shape elsewhere in the peak, shown in Figure 5. The analyte in question is in the library being used, but it is not being searched for in all the time slices in which the analyte is present.

Figure 3. Unexpectedly high Log Chi2 value for time slice at the beginning of a peak

Figure 5. Absorbance spectrum of time slice to the immediate right of the one shown in Figure 4. There is absorbance saturation, but the blue fit area looks to fit the absorbance spectrum.

This issue could be caused by a problem with the retention index file, but it could also be caused by a problem with time range limitations in our Analytes to Include (ATI) list.  The ATI list can be used to add individual compounds to the analysis, or to specify a time window for when to look for a particular analyte.  The analysis shown here was an unmodified D8071 PIONA analysis, and it did not have any analytes designated in the ATI list.  Checking the ATI list in this situation confirms that we have no analytes selected, so the source of the problem is likely the RI file. For further confirmation of this we will check our RI values and verify them in VUVision™ Software.

Figure 6. Analytes to Include tab where analyte time range limits would be shown.

Finding analysis RI times

VUV Analyze reports, whether pdf or plain text, always include the RI file information with the retention times used in the analysis. An example from the pdf report is shown in Figure 7.

Figure 7. Example RI file values from a D8071 PDF report.

Examining the file in VUVision

We next load the file in the Data View window of VUVision.   Double clicking on the first few peaks of the Peak List we find several analytes from our RI file, and the Retention Indexes for all of them have shifted a bit from what was seen in the RI file.  For example, the isopentane was predicted at 2.80 minutes from the above RI file, but here we see it eluting at 2.77 minutes.  Shifts of 0.03 minutes or more should be investigated to examine possible causes.

Figure 8. Iso-pentane appears in both the RI file and the run file being analyzed. The retention time is slightly off what was seen in the RI file.

Possible Causes of retention time shifts

The only setting on a VGA that will cause any change in retention time is the makeup gas pressure. Increased makeup gas results in increased backpressure on the column and thus increased retention times. A large change in makeup gas pressure can affect retention times accordingly.  If the wrong acquisition method was used (for example the acquisition method for ASTM D8267 jet analysis was used with an ASTM D8071 PIONA sample), the difference in the makeup gas pressures between the two methods might be enough to shift retention times.

Other causes of retention time shift come from the GC. The GC method should be checked to ensure that all settings are correct.  Using the wrong column dimensions on the GC is a common source of problems.  Leaks close to the inlet, such as holes in the septum, will result in analytes taking longer to elute.  Column aging can also cause slight changes in retention times.  If GC problems have been ruled out and the VGA acquisition method is correct, the VUV-RT should be rerun to obtain a new RI file and production file.

This post covered the problem of VUV Analyze only missing parts of peaks.  If the analysis is missing entire peaks for reasons not relating to retention time issues, it is possible an analyte is not in the library being used for analysis.  For discussion of this issue and how to resolve it, see GETTING TO KNOW VUV ANALYZE™, PART 1.