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Normal-Phase vs Reversed-Phase HPLC
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One of the most important choices in chromatography method development is deciding between Normal-Phase (NP) and Reversed-Phase (RP) HPLC. The following comparison outlines the main differences:

Normal-Phase HPLC

The stationary phase is more polar than the mobile phase.

Common column types include Silica, Amino (NH2), Diol, and Cyano (CN).

The mobile phase usually consists of non-polar solvents such as hexane or ethyl acetate.

Polar compounds are retained for a longer time.

Reversed-Phase HPLC

The stationary phase is less polar than the mobile phase.

Typical columns include ODS (C18), C8, C4, Phenyl, and Cyano (CN).

The mobile phase often contains polar solvents such as water or acetonitrile.

Non-polar compounds are retained for a longer time.

The retention order of analytes is essentially reversed when switching between NP and RP systems—this principle lies at the core of chromatographic separation.

Choosing the appropriate phase depends on factors such as analyte polarity, sample solubility, and the desired separation outcome.

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Frequently Asked Questions: HPLC Analysis & Chromatography

High-Performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify each component in a mixture. It relies on a pump to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.

Column efficiency is typically measured by the number of Theoretical Plates ($N$). The most common formula is $N = 16 \times (t_r / W)^2$, where $t_r$ is the retention time and $W$ is the peak width at the base. A higher number of theoretical plates indicates a sharper peak and better analytical separation. You can calculate this instantly using our Theoretical Plates Calculator.

The ICH (International Council for Harmonisation) Q2(R1) guidelines mandate specific validation parameters for HPLC methods. These include assessing Accuracy, Precision (Repeatability and Intermediate Precision), Specificity, Detection Limit (LOD), Quantitation Limit (LOQ), Linearity, and Range. Our calculators are designed specifically to help analysts easily compute these linearity, LOD/LOQ, and %RSD parameters in compliance with ICH standards.
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