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The What, When, and How of Peak Integration: Part 2. When.

Not every method will result in peaks being baseline resolved, or have peaks all the same size.  In the second installment of this series we will define some of the situations which most commonly occur and result in difficult integration.

When Peaks Are Difficult to Integrate?

Errors with integration can be the result of poor method development (e.g. sample preparation, chromatographic separation, incorrect detector settings) or an instrument fault (e.g. poor autosampler injection precision).
Peaks will be difficult to integrate, and consistently and accurately quantitate under the following circumstances (Figure 1):

  • On a rising, falling, or drifting baseline
  • Minor peaks on the tail of major ones
  • Tailing or fronting peaks
  • Peaks of very different heights 
  • Partially resolved peaks
  • Peaks of different sizes and ratios

Figure 1: Difficult to integrate peaks.

In order to avoid some of the situations detailed above we should look to try and (where possible) improve resolution, obtain a stable baseline, avoid peak tailing or large solvent peaks, and assess if our method needs to be altered when dealing with samples which have analytes in very different concentrations (i.e. produce peaks of very different area, ratio, and/or height).

Improve Resolution

Resolution is a function of selectivity, efficiency, and retention.  Making improvements to any of these parameters will positively impact on resolution.

For HPLC, retention and selectivity will both be altered by changing the organic solvent, mobile phase pH, solvent strength and additives, stationary phase chemistry, and temperature.  Efficiency can be increased by increasing the column length, reducing the column internal diameter, or decreasing the particle size.  It is better to use a smaller diameter packing than increase the column length, which will increase analysis time.  However, a decrease in particle size will result in an increase in system backpressure.  The use of smaller particles and narrower column internal diameter both require minimized extra column dead volume in order to avoid efficiency losses.

The most effective way to alter the retention factor in GC is to adjust the temperature of the carrier gas.  Other variables which also affect retention and selectivity are the oven temperature ramp rate, chemical nature of the stationary phase, and the ratio between the film thickness and column diameter (phase ratio, β).  Column dimensions (internal diameter, film thickness, and length) are used to optimize efficiency.

 

Obtain a Stable Baseline

Baselines should be flat and stable throughout a chromatographic analysis, yet there are numerous baseline related problems such as drifting, cycling, noise, and spikes which can be the result of both method and instrument problems.  

For example, unstable baselines in HPLC can be the result of temperature fluctuations, normal re-equilibration during gradient analysis (re-equilibration time will need to be assessed on a method by method basis, particularly when working with ion-exchange, HILIC, or ion pair applications), mobile phase variations (solvent evaporation or variation in additive concentration during gradient analysis), flow rate variation, leaking pump or piston seal, or a faulty check valve.

In GC column bleed (all columns bleed, although more polar and thicker stationary phases bleed more), a column which is not properly conditioned, variable carrier gas flow, column contamination,  inlet/liner contamination, and detectors not properly equilibrated can all lead to baseline problems.

Avoid Peak Tailing or Fronting

Peak tailing can be particularly insidious when working with ionizable, polar, or active compounds as these can interact with any active sites (usually acidic silanol species) within the HPLC or GC system.

To avoid peak tailing in HPLC analyses ensure pH control is used for ionizable analytes or use type II/III silica which has less surface silanol groups.  Tailing peaks can also be an indication of column contamination, blocked/contaminated column end frits, sample diluent/eluent mismatch, or that the sample concentration or injection volume is too high.

For GC applications peak tailing can be minimized by using columns which are designated GC-MS or highly-inert (these exhibit low bleed and low silanol activity) and using deactivated inlet consumables.  Again peak tailing can be a clear indication of problems such as column degradation, inlet/liner contamination, an incorrect column cut, or poor column installation.  In regards to detectors low makeup gas (FID and ECD detectors), MS source or transfer line not hot enough, or the column outlet end extending through the detector jet into flame (FID) can all result in tailing peaks.

In both HPLC and GC peak fronting is usually a sign of column overload, this can be remedied by reducing the concentration or injection volume of the sample.

Avoid Large Solvent Peak

Really this a GC concern, especially when carrying out splitless injection, if the splitless time is too long then the solvent peak will show excessive tailing.  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.  Typical splitless times are in the region 20-90 seconds.

Some of the problems identified above may be unavoidable, therefore, in the third and final installment of this series we will discuss the use of different integration settings and how they can be applied to difficult to integrate peaks.

 

Further Reading

Integration Problems »

Integration Errors in Chromatographic Analysis, Part I: Peaks of Approximately Equal Size »

Integration Errors in Chromatographic Analysis, Part II: Large Peak Size Ratios »

 

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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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