Retention of Guanidine Compounds Analyzed by HPLC- AppNote
June 24, 2026
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Date: 24-June-2026 Last Updated: 24-June-2026
Separation of Highly Polar Guanidine Compounds
This method demonstrates effective retention, resolution, and peak shape for highly polar, basic compounds using HILIC on a Cogent Diol column, highlighting its suitability for challenging analytes.
While alternative approaches such as ion-pair reversed phase or mixed-mode chromatography can provide retention of these highly polar bases, this HILIC method offers a simpler, MS‑friendly solution with strong retention and balanced selectivity for all analytes.
Fig 1. Blank signal has been subtracted from the sample chromatgram to correct for baseline drift of this low UV wavelength. This removes background noise and drift caused by the detector.
Peaks:
1. Cyanoguanidine
2. Guanidine
3. Melamine
4. Metformin
Method Conditions
Column:
Cogent Diol™, 4 µm, 100 Å
Catalog No.: 40060-25P
Dimensions: 4.6 x 250 mm
Mobile Phase:
A: DI Water + 0.1% formic acid
B: Acetonitrile + 0.1% formic acid
Gradient:
Catalog No.: 40060-25P
Dimensions: 4.6 x 250 mm
Mobile Phase:
A: DI Water + 0.1% formic acid
B: Acetonitrile + 0.1% formic acid
Gradient:
| Time (minutes) | %B |
|---|---|
| 0 | 75 |
| 1 | 75 |
| 10 | 65 |
| 10.5 | 75 |
| 20 | END |
Injection Volume: 1 uL
Detection: 195 nm
Diluent: 50:50 ACN:DI Water + 0.1% formic acid
Final working concentrations: Cyanoguanidine 0.0310 mg/mL (31.0 µg/mL); Guanidine 0.414 mg/mL (414 µg/mL); Melamine 0.0276 mg/mL (27.6 µg/mL); Metformin 0.138 mg/mL (138 µg/mL).
Using phenanthrene (t₀ = 2.66 min) to estimate void time, retention factors (k) listed below.
- Cyanoguanidine: 3.40 min, k* ≈ 0.28
- Guanidine: 11.75 min, k* ≈ 3.42
- Melamine: 12.34 min, k* ≈ 3.64
- Metformin: 13.32 min, k* ≈ 4.01