Chromatography Theory: Peak Identification & Quantification

Chromatography Theory. Peak Integration

Overview

This chapter closes out Integration Theory with two critical steps: Peak Identification and Peak Quantification, chromatography peak identification. You’ll learn how Chromperfect uses calibration files, search windows, and optional retention-time referencing to identify components—even in crowded regions—and how final amounts are calculated reliably with the right corrections.

Quick context: Peak detection and baseline integration are already complete at this stage.

Area Reject Threshold: Filtering the Noise

Chromperfect provides a second line of defense against spurious peaks via the Area Reject Threshold defined in the calibration file.

• Peaks below this threshold stay in the peak list but are excluded from totals, labels, and reports.

• With no calibration file, the threshold defaults to zero (all positive-area peaks pass).

• Rare negative-area peaks (often from manual baselines) can be excluded by keeping the threshold ≥ 0.

Tip: If you must include negative areas (unusual), set the threshold to a sufficiently negative value.

Retention-Time Referencing: Keep Windows on Target

When peaks shift proportionally (e.g., slight flow-rate changes), retention-time referencing moves dependent search windows to track the reference peak.

• Choose a reference component near the target group, ideally in a clean region.

• Chromperfect selects the largest-area candidate within the reference window (subject to its own area-reject threshold).

• Use multiple references (early/mid/late) if drift isn’t uniform across the run.

• If the reference is absent or too small, windows do not move (protects from mis-assignment).

Peak Identification: One Peak ↔ One Component

Each component in the calibration has a search window (expected time + width). Identification rules:

• If ≥1 peak is inside the window, the center-most peak is assigned.

• Identification is one-to-one: no component gets two peaks, and no peak serves two components.

• Overlapping windows are discouraged; if they occur, Chromperfect resolves conflicts by favoring the narrower (or earlier, if tied) window.

Best practice: Tighten windows with retention-time referencing to avoid overlaps.

Multi-Peak & Nested Windows: Real-World Flexibility

Some applications need a sum of all peaks in a region (multi-peak) while excluding specific aromatics that are quantified individually. Chromperfect handles both:

• Multi-Peak window: Sums all legitimate peak responses under the window (area or height) to produce Window Response and Window Amount, while still reporting the “center-most” single-peak values if desired.

• Nested windows (e.g., C6+ fraction): Smaller (often single-peak) windows are evaluated first, staking a claim on their peaks. Larger windows then sum the remaining peaks in their range. This yields correct totals without double-counting and keeps benzene/toluene/xylenes separate from the broad alkane fraction.

Quantification: Area vs. Height

Chromperfect supports quantification by area or height:

• Area is often preferred with mass-sensitive detectors (FID/TCD) and well-resolved peaks.

• Height can be more stable when peaks are poorly resolved or S/N is low, and with concentration-sensitive detectors (e.g., UV) where flow variations matter.

  
  

Calibration Curves

• External or internal standard approaches are supported.

• Curves can be linear/non-linear, through the origin or with intercepts.

• Amounts are first computed as Raw Amounts from the calibration curve.

Essential Corrections (Applied After the Curve)

Chromperfect applies independent correction factors to produce the Corrected Amount:

1. Dilution Factor (DF):Corrected = Raw × DF Use DF for dilutions before spiking an internal standard (post-spike dilutions cancel in ratios).

2. Injection Volume (IV):Corrected = Raw × (Standard IV / Actual IV)Set Standard IV in the calibration; Actual IV in the raw file. Note: For internal-standard methods, IV variation typically cancels in the ratio—no separate IV correction applied.

3. Sample Weight (SW):Corrected = Raw × (Standard SW / Actual SW)Use this to report, e.g., mg per g, mg per 100 g, or mg per kg by picking a suitable Standard SW (1, 100, or 1,000,000 mg).

Combined equation: Corrected Amount = Raw × DF × (Std IV / Act IV) × (Std SW / Act SW)

Default Component: Quantify Unassigned Peaks

Unidentified peaks (outside all windows) can still be quantified using a Default Component:

• Set the default to a specific component or to the previous/next/nearest.

• Internal-standard group and limits follow the default component.

• Be aware that “nearest” can flip with small RT variations for peaks between two windows.

Window Amount & Response

For each component, Chromperfect also computes:

• Window Response: sum of all legitimate peak responses within the window.

• Window Amount: the corresponding amount from the calibration curve.

These are optional but invaluable in formatted reports for multi-peak/nested strategies.

Normalized Quantification (Area%, Height%, Amount%)

When results must sum to 100%:

• Area% / Height% require no calibration; best when detector response is uniform across analytes.

• Amount% uses calibrated amounts (recommended when response factors differ).

Internal-standard peaks and sub-threshold peaks are excluded from the totals by design.

Practical Setup Checklist (Chromperfect)

•  Define Area Reject Threshold in the calibration to suppress noise.

•  Pick reference peaks and enable retention-time referencing if RT jitter exists.

•  Avoid overlapping windows; prefer tighter windows with referencing.

•  Use Multi-Peak or Nested windows for fraction sums (e.g., C6+) while keeping aromatics individual.

•  Set Standard IV and Standard SW in the calibration; enter Actual IV/SW per run in raw files.

•  Configure a Default Component if you want unassigned peaks quantified.

•  Choose Area%/Height%/Amount% normalization to suit your detector and reporting needs.

Chromatography peak identification - Support

Need help configuring windows, references, or formatted reports for regulated workflows? Get in touch—we can review your calibration and reporting setup and recommend best practices for Chromperfect 10.