HPLC Explained

How High-Performance Liquid
Chromatography Works

A plain-language guide to the instrument, the method, and exactly how ANALITIKA derives a purity percentage from your sample — with no prior chemistry knowledge required.

What Is HPLC?

Separating compounds to identify and measure them

HPLC stands for High-Performance Liquid Chromatography. In simple terms, it is a technique for separating the individual components of a liquid mixture so each one can be identified and measured independently.

Think of it like a race. You dissolve your sample in a liquid (the mobile phase) and push it through a tightly packed column at very high pressure. Different compounds travel through the column at different speeds — some stick more to the packing material, some less. By the time they reach the end of the column, they have separated into individual bands, each arriving at a different time.

A detector at the end of the column measures each compound as it passes. The result is a chromatogram — a graph showing which compounds were present and how much of each.

Shimadzu Nexera HPLC System

Shimadzu Nexera LC-40 — the UHPLC system used at ANALITIKA

The Instrument

What each module does

A modern UHPLC system like the Shimadzu Nexera is made up of several connected modules, each with a specific role in the analysis.

LC-40 Series

Solvent Delivery Pump

Pushes the mobile phase (typically water + acetonitrile) through the column at precisely controlled flow rates and pressures up to 130 MPa. The pressure is what gives HPLC its speed and resolution.

SIL-40C Series

Autosampler

Automatically injects a precise, reproducible volume of your dissolved sample into the mobile phase stream. Carryover between samples is less than 0.0003% — critical for accurate back-to-back testing.

CTO-40 Series

Column Oven

Holds the analytical column at a precise, stable temperature throughout the run. Temperature affects separation — keeping it constant ensures every run is directly comparable to the last.

Reversed-Phase C18

Analytical Column

The heart of the system. A stainless steel tube packed with microscopic C18-bonded silica particles. Compounds interact differently with this surface, which is what causes them to separate.

SPD-M40

PDA Detector

A Photodiode Array detector that shines UV light through the column eluent and measures how much is absorbed at 1,024 different wavelengths simultaneously — detecting compounds as they exit the column.

SCL-40

System Controller

A touchscreen controller and data system (LabSolutions) that orchestrates every module, captures the raw signal, integrates peak areas, and produces the chromatogram data used in your report.

HPLC Column Separation Diagram

Compounds separating as they travel through the reversed-phase column

The Column

How reversed-phase separation works

In reversed-phase HPLC, the column is packed with C18-bonded silica particles — essentially microscopic beads coated in a waxy, non-polar surface. The mobile phase flowing through is a polar mixture of water and organic solvent (typically acetonitrile).

When your sample enters the column, each compound within it partitions between the mobile phase and the C18 surface based on its own polarity. More non-polar compounds stick to the waxy surface longer, eluting (exiting) later. More polar compounds prefer the aqueous mobile phase and elute earlier.

By the time a compound reaches the detector, it has been physically separated in time from everything else in the sample — so each compound produces its own distinct signal.

The Chromatogram

Reading the output

The chromatogram is the raw output of the HPLC run — a graph with time on the horizontal axis and UV absorbance on the vertical axis. Each peak represents one compound that passed through the detector.

Retention Time
The time it took a compound to travel through the column. Each compound has a characteristic retention time under a given set of conditions, which helps confirm identity.
Peak Height & Area
The height and area of a peak are proportional to the amount of that compound present. Larger peaks = more material.
Baseline
The flat line between peaks representing zero absorbance. Clean separation between peaks and a stable baseline are signs of a well-developed method.
HPLC Chromatogram Example

Example chromatogram: one main peak (98.7% purity) with two small impurity peaks

The Calculation

How purity percentage is derived

Purity is calculated using the area percentage method — one of the most widely accepted approaches in analytical chemistry.

Purity Formula
Purity (%) = Area of Target Peak ÷ Sum of All Peak Areas × 100
01

Integrate all peaks

The LabSolutions software automatically calculates the area under every peak in the chromatogram, including the main compound and any impurities.

02

Sum all areas

All peak areas are added together to get the total detected material in the sample. This is the denominator in the purity calculation.

03

Divide and express as %

The area of the target compound's peak is divided by the total area, then multiplied by 100. The result is the chromatographic purity percentage reported on your COA.

Your Results

What your Certificate of Analysis contains

Every COA issued by ANALITIKA is a complete, traceable document. Here is what each field means.

Compound Name & Lot / Batch
The material as you identified it when submitting the sample, together with the lot or batch number on the vial, so the report can be matched to exactly what was tested.
Purity (%)
The chromatographic purity — the percentage of the detected signal that belongs to the target compound. Calculated using the area percentage method described above.
Measured Mass (mg)
The actual measured mass of compound content in the sample, calculated by comparing the peak area against a certified reference standard of known concentration.
Fill Accuracy (%)
How close the measured mass is to the label claim. A fill accuracy of 100% means the vial contains exactly what it claims. Values outside 95–105% may indicate under- or over-filling.
Retention Time (min)
The time the compound took to travel through the column. This is a qualitative identity indicator — compounds have characteristic retention times under a given method.
Chromatogram
The actual raw output of the HPLC run, included on every COA so you and your customers can see the full separation — not just the final number.
Verification Code & QR
A unique code and scannable link that lets anyone — your customers, distributors, or regulators — verify the COA is genuine and unaltered by checking it against our records.

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