TLC to Gradient Chromatography Separation Simulator

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Theoretical Introduction to Gradient Chromatography Estimation from TLC

This simulator predicts how compounds will separate in a gradient chromatography column based on their Thin-Layer Chromatography (TLC) behavior. It's a useful tool for optimizing purification methods, especially for flash chromatography and preparative HPLC.

1. Rf and k' Correlation

The **retardation factor (Rf)** from TLC directly relates to the **retention factor (k' or capacity factor)** in column chromatography. A higher k' means stronger retention.

k' = (1 - Rf) / Rf

This equation bridges TLC observations with column retention.

2. Modeling Retention in a Gradient: log(k') vs. Solvent Strength

In gradient elution, the mobile phase composition constantly changes, so a compound's k' isn't constant. For a binary solvent system (Solvent A and Solvent B), the logarithm of the retention factor (log(k')) typically relates linearly to the **volume fraction (phi)** of the strong solvent (Solvent B):

log(k') = log(k'0) - S * phi

Here:

The simulator determines k'0 and S for each compound using **linear regression** on data from multiple TLC runs at different Solvent B percentages. You'll need at least two such data points.

3. Simulating Gradient Elution in Column Volumes (CV)

The simulator predicts compound behavior under a linear gradient using **Column Volumes (CV)**. One CV equals the column's dead volume (Vm).

The process uses a **step-by-step numerical integration**:

  1. The gradient (initial %B to final %B over a specified number of gradient CVs) is divided into small steps.
  2. For each step, the average solvent composition (phi) is determined.
  3. The instantaneous k' for each compound is calculated using its k'0 and S values.
  4. The compound's "migration progress" within that step is calculated. A compound's velocity is proportional to 1 / (1 + k').
  5. This progress accumulates. A compound "elutes" when its accumulated progress equals 1 (it has traversed one dead volume).
  6. The **total elution volume (Ve) in CV** is the sum of the gradient CVs elapsed until elution, plus 1 CV for the initial dead volume.

4. Estimating Resolution (Rs)

**Resolution (Rs)** quantifies peak separation:

Rs = 2 * (t_R2 - t_R1) / (w1 + w2)

Where t_R are elution volumes and w are peak widths. Peak widths are estimated based on the **number of theoretical plates (N)** of the column:

w_CV = 4 * (Ve_CV / sqrt(N))

Calculating Rs helps assess separation success.

5. Normal Phase vs. Reverse Phase Considerations

The general principles apply to both modes, but Solvent B's nature differs:

The model consistently interprets Solvent B as the "stronger" eluent, leading to decreased retention as its concentration increases. A warning appears if the calculated S parameter suggests otherwise, indicating potential data inconsistency.

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1. TLC Data (At least 2 TLCs with different % Solvent B)

Enter **RF values between 0.01 and 0.99** (excluding 0 and 1 to avoid infinite or zero k'). Enter at least **2 TLCs** with different Solvent B percentages for a reliable gradient estimation.

2. Linear Gradient Parameters

**Gradient Duration (CV)**: The number of **column dead volumes** (Vm) that are eluted during the solvent composition change. E.g., a "10 CV" gradient means the solvent composition changes completely while 10 dead volumes pass through the column.

**N** influences peak width and thus resolution. Typical values: 500-5000 for flash chromatography, 5000-50000 for HPLC.

3. Gradient Simulation Results

The graph shows the gradient profile (%B vs. CV) and the estimated peak positions of your compounds. The X-axis represents the total column volumes (CV) eluted.