System State ● IDLE

Pressure 0.0 bar

Flow Rate 1.00 mL/min Flow Stable ✓

UV Wavelength 254 nm UV Lamp ● ON

Column Oven 25.0 °C

Real-Time Chromatogram Signal

IDLE

📈

Your chromatogram will appear here

Tap ▶ Run Experiment for 1-tap automated run!

            Sample: Paracetamol
            Flow: 1.00 mL/min
            λ: 254 nm
            Temp: 25°C
            Run: 5.0 min
          

💡 Live Separation Guide & Theory

STATUS: READY

Select your Sample and Mobile Phase %B on the right, then tap ▶ Start Separation to watch analyte molecules travel through the reversed-phase $C_{18}$ column.

HPLC Operations

🟢 Beginner Mode

⚡ Quick-Start Presets:

1️⃣ SELECT SAMPLE ANALYTE:

              2️⃣ SET CONDITIONS:
            

Mobile Phase %B (Organic Solvent): 40%

◀ Better Separation (Slower) Faster Elution ▶

⏱️ Period of Operation (Run Time): 5.0 min

Flow Rate: 1.0 mL/min

Optimal Van Deemter velocity: ~1.0 mL/min

Column Temperature: 25°C

BUFFER SYSTEM

UV Wavelength (λ): 254 nm

              3️⃣ EXECUTE SEPARATION:
            

MANUAL HARDWARE CONTROLS:

SIMULATION SPEED:

USP <621> System Suitability & Peak Integration

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Ready for Peak Evaluation

Execute a run on Tab 1 to record analyte retention times ($t_R$), calculate theoretical plates ($N$), tailing factor ($T$), and resolution ($R_s$).

🎓 HPLC Method Development & Separation Masterclass

Master reversed-phase C18 partitioning, Van Deemter column band broadening kinetics, and USP <621> chromatographic resolution equations.

🔬 HPLC High-Pressure Flow Schematic

Loading diagram…

📊 On-Line Photodiode Array (PDA) Spectrum

📊 Interactive Van Deemter Column Efficiency Calculator (H vs u)

The Van Deemter equation $H = A + \frac{B}{u} + C \cdot u$ describes plate height ($H$) as a function of mobile phase linear velocity ($u$):

Flow Rate (F): 1.00 mL/min

Plate Height (H)

12.4 µm

Theoretical Plates (N)

12,100 plates

💡 Sub-2µm particles dramatically reduce the C-term (resistance to mass transfer), allowing 2–3x faster flow rates without losing chromatographic efficiency.

Plate Height H vs Linear Velocity u

0.2 mL/min (B/u Eddy) u_opt (Minimum H) 3.0 mL/min (C·u Resistance)

🎯 Interactive Purnell Resolution Masterclass (R_s)

The fundamental resolution equation links peak separation ($R_s$) to the three pillars of chromatography: R_s = (√N / 4) · ((α - 1) / α) · (k₂' / (1 + k₂'))

1. Efficiency (Plate Count N): N = 8,000

2. Selectivity (Separation Factor α): α = 1.15

3. Retention Factor (Capacity k₂'): k₂' = 3.0

Calculated USP Resolution

R_s = 1.94

USP PASS (≥1.5)

Live 2-Peak Chromatogram Simulation

Peak 1 (tR1) Valley Baseline Peak 2 (tR2)

📐 USP <621> Peak Symmetry & Diagnostic Defect Compendium

Pharmacopoeial assays enforce strict system suitability criteria. Deformed peaks indicate hardware failure, chemical overloading, or secondary interactions.

Ideal Gaussian Peak

0.95 ≤ T ≤ 1.20

Symmetric band shape indicating linear adsorption isotherm and homogenous stationary phase packing.

Peak Tailing (T > 1.5)

T = W₀.₀₅ / 2f > 1.5

Caused by unreacted acidic silanol sites binding basic amine drugs, or column void formation. Fix: Add triethylamine / buffer at low pH.

Peak Fronting (T < 0.9)

T < 0.85 (Shark Fin)

Caused by column mass overload or dissolving sample in 100% organic solvent stronger than mobile phase. Fix: Dilute sample or match mobile phase.

Peak Splitting / Doublet

Dual Apex / Shoulder

Caused by partially blocked inlet frit creating dual flow paths, or sample precipitation on column head. Fix: Backflush column or replace guard.

🌳 Method Development Decision Tree & Linear Solvent Strength (LSS) Model

Under the Linear Solvent Strength (LSS) model, analyte retention factor ($k'$) drops exponentially as organic modifier volume fraction ($\phi$) increases: $\log k' = \log k_w - S\phi$.

Step 1: Analyte Ionization Assessment

• Acidic Drug (e.g. Aspirin, Ibuprofen, pKa ~3.5–4.4): Lower buffer pH to 2.5 (suppresses ionization, retains strongly on C18).
• Basic Drug (e.g. Quinine, Metronidazole): Use pH 2.0 or pH 7.0 with ion-pairing modifier to prevent silanol tailing.

Step 2: Isocratic vs Gradient Elution

• Δk' < 20: Use Isocratic Elution (e.g. 50:50 ACN/Water) for simple tablets. Zero column re-equilibration time.
• Δk' > 20: Use Organic Gradient Ramp (e.g. 10% → 90% B over 10 min) to elute hydrophobic impurities in a single run.

HPLC High-Performance Liquid Chromatograph Simulation