HPLC-CAD Piperidine Sample Test Report

1. Instruments and Materials

Instrument: Agilent 1200 liquid chromatograph with an Instrumax CADetector a1 Charged Aerosol Detector (CAD).

Reagents: pure water, acetonitrile (chromatography grade) and trifluoroacetic acid (analytical grade).

Samples: standards and piperidine sample supplied by the user.

2. Methods and Results

2.1 Chromatographic Conditions

Column: Phenomenex Kinetex® XB-C18, 5 μm, 4.6 × 250 mm.

Column temperature: 35 °C. Flow rate: 1.0 mL/min. Injection volume: 10 μL.

Mobile phase A: 0.1% TFA aqueous solution. Mobile phase B: 0.1% TFA acetonitrile solution.

Gradient conditions:

CAD conditions: CADetector a1; drift tube 50 °C; atomizing gas 3 L/min; corona current 1 μA; corona gas 1 L/min; gain 0.2; RF 2.0.

2.2 Sample Information

Blank solvent, 15 µg/mL standard, test sample, and 10%, 20%, 30%, 50%, 80%, 120%, 150% and 200% standard levels were prepared. The corresponding concentrations were 1.5, 3.0, 4.5, 7.5, 12.0, 18.0, 22.5 and 30.0 µg/mL.

2.3 Experimental Results

2.3.1 Specificity

The blank solvent did not interfere with the target piperidine peak, showing good specificity.

2.3.2 Sensitivity

For the 10% concentration-level sample, the signal-to-noise ratio calculated by the Agilent CDS P2P method was 43.51, meeting the sensitivity requirement.

Figure 1. Blank solvent / diluent chromatogram.

Figure 2. 10% concentration-level standard and S/N result.

2.3.3 Linearity

Nine concentration-level standards were injected once each, and the standard was also injected at different injection volumes. Linear equations were generated from concentration versus peak area and injection volume versus peak area. Concentration linearity gave R²=0.9980, and injection-volume linearity gave R²=0.9995, indicating good linearity.

2.3.4 Repeatability

The standard was injected six consecutive times, and retention time, peak area and peak height RSD% were calculated. Retention-time RSD was 0.184%, peak-area RSD was 1.38%, and peak-height RSD was 1.65%; all were below 2.0%, showing good repeatability.

2.3.5 Sample Test

A 10 μL piperidine sample injection gave a clear target peak with a peak area of 23.542. The concentration calculated from the linear calibration curve was 2.9890 µg/mL.

Figure 3. Repeatability chromatogram for six standard injections.

Figure 4. Sample chromatogram, full scale.

Figure 5. Sample chromatogram, enlarged view.

1. Experimental Discussion:
2. The reference method uses a Waters T3 column. Because of available conditions, this test screened other C18 columns and finally selected a Phenomenex Kinetex XB-C18 column. The method basically met the experimental requirements, although the baseline near the target peak was slightly uneven.
3. The experiment evaluated CAD suitability for the sample from specificity, sensitivity, linearity and repeatability. The results show good CAD sensitivity, linearity and repeatability and meet the experimental needs.