Triggering Fractionation

Preparative Chromatography Course

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1 - Fundamentals of Preparative Chromatography 

2 - Preparative Workflow 

3 - Preparative Chromatography Instrumentation 

4 - Key Considerations: Part 1 

5 - Key Considerations: Part 2 

6 - Column Selection 

7 - Automated Preparative Purification Instrumentation - UFPLC 

8 - Automatic Analytical to Preparative Scale Up

9 - Triggering Fractionation

10 - Preparative SFC

11 - Course Summary & Test

preparative chromatography online course

Triggering Fractionation 

Welcome to the penultimate course unit, based on the different triggers which are most used to begin collecting fractions in preparative chromatography. Through this unit, you will gain an understanding of the difference between the different triggers which can be used to commence fractionation, including UV, time and mass directed.   

Types of Triggers

Fraction Collection Trigger Methods
Types of Trigger How It Works Advantages Limitations
Time Based Start fraction collection after a fixed time from injection or from a reference event. 
  • Simple to implement and robust. 
  • Useful when retention times are very reproducible.
  • Sensitive to retention time drift caused by aging columns and other variables.
  • Not suitable for overlapping peaks or complex mixtures.
UV Detector Threshold  Start when absorbance at one or more wavelengths exceeds a preset threshold (absolute or relative). 
  • Good for peaks with strong, unique chromophores.
  • Good for peaks with strong, unique chromophores.
  • Not effective for UV inactive compounds.
  • Cannot be used for compounds with similar chromophores (i.e. isomers).
  • Baseline noise, drift or coeluting impurities at the same wavelength can cause false triggers.
Mass Directed Uses an MS signal based on a single m/z or extracted ion chromatogram to trigger collection when target ion exceeds a threshold.
  • High specificity and sensitivity, which is ideal for coeluting species, low abundance targets.
  • Can trigger based on multiple ions or ion ratios for confirmatory specificity.
  • Higher cost and complexity, requires compatibility between MS and fraction collector including split flow, and make up solvent.  
  • Matrix ion suppression can affect intensity. 
Conductivity or refractive index Start when conductivity or RI changes above a certain threshold.  Useful for ionic species or non-UV-active polar compounds. 
  • RI detectors are less sensitive and respond to mobile phase composition changes, which prevents gradients being used. 
  • Conductivity works for ionic analytes, but mobile phase buffer salt concentration can mask signals.
Fluorescence or Evaporative Light Scattering Detector (ELSD) Triggered based on properties of the analyte (i.e. fluorescence detector thresholds or the scattered light profile).
  • ELSD is a universal detector.
  • High sensitivity and selectivity for fluorescent compounds.
  • Non-linear response, sensitivity to mobile phase changes. 
  • Requires analytes to be fluorescent or derivatised.

  

Considerations for Trigger Selection 

  • Nature of the analyte – are they UV active, volatile, or possess some other characteristic which would determine which trigger.  
  • Matrices – Are the samples in complex matrices or contain a vast number of impurities?  
  • Method type – certain techniques, such as RI can only be used with isocratic conditions.  
  • Required purity and yield   
  • Throughput 
  • Reproducibility 
  • System compatibility and cost 

 

UV vs MS Directed Trigger

Depending on the objective of the experiment, HPLC can be divided into two main categories: analytical HPLC, used for identification and quantification, and preparative HPLC, used for isolation and purification.

Chromatogram showing UV-triggered fraction collection around the hydrocortisone peak, highlighting salicylic acid and impurity peaks.

 

Let’s look at an application for the collection of hydrocortisones. The separation shown here used a 150 x 20 mm ShimPack Scepter C18 using a low pH gradient with acetonitrile. The resulting chromatogram had resolved the main impurity from hydrocortisone, however, there were two smaller impurities which coeluted in the front and the tail of the peak of interest when the load increased. With a UV triggered event, these two impurities were taken into the fraction collection, which compromised the purity of the fraction.  

Chromatograms showing UV and MS trigger signals used to define hydrocortisone fraction collection intervals while excluding impurity peaks.

Figure 2.  UV and MS Triggered Fraction Collection of Hydrocortisone

Table summarising preparative LC and MS method parameters, including mobile phase, column, flow rates, detection settings and ionisation conditions for hydrocortisone analysis.

Table 1.  Preparative LC–MS Conditions for Hydrocortisone Fraction Collection
Overlay chromatograms of reinjected hydrocortisone fractions collected using UV-only and UV–MS triggers, showing reduced impurity peaks with UV–MS triggering.

 

This figure shows the chromatogram obtained when a fractionated hydrocortisone was reinjected into the analytical flow path, as well as the chromatogram of a standard mixture, as a reference for calculating recovery. The blue trace is a solely UV triggered response, whilst the red is a combination of UV and MS trigger. 

Table comparing purity and recovery rates of hydrocortisone fractions collected using UV-only and UV–MS triggers, showing high purity and recovery for both methods.

 

It can be easily observed that the impurities are reduced when complementary techniques are employed to trigger the fraction collection. In this example, the recovery for UV trigger alone was 99.7%, which is excellent, however, with a UV / MS trigger, this increased to 100.0%. 

 

 

 

 

 

 

 

That concludes our penultimate module, where we have described different triggers and considerations which need to be addressed for why you’d select certain approaches. We have also highlighted the power of employing mass directed triggers in conjunction with UV threshold triggers. 

Best regards, 
Your Shimadzu Chromatography Team 

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