[Readers Insight] Why is the First Injection in Chromatographic Analysis Unreliable?

This article is written by Welch's contract writer Chromatography Mound. The content of the article presents a point of view from the author solely.

A junior analyst once texted me and asked, “Professor. I know we always discard the data from the first injection, or use a blank run as the first injection. But I want to know the exact why the first injection is unreliable. Can you explain to me?”

This question is fair. In laboratories, discarding the first injection from quantitation is a universally acknowledged “chemistry”. Even if you are using a top-tier instrument or have equilibrated the column for a full two hours, daring to use the first injection for quantitative reporting will likely result in a wildly off‑base response value. Therefore, the first injection should be either a blank run or a high‑concentration sample as a “primer”.

But why does this phenomenon occur? It is by no means magic, but a "cold‑start" behavior of chromatographic instruments. What follows in this article is my explanation to the reasons behind this behavior at several microscopic levels: column, autosampler, and equilibrium.

Unsaturated active sites is the most common culprit behind a low response on the first injection. While HPLC columns are meticulously manufactured and tested before shipment, sites that are hungry for interaction still exist in the flow path, including the stainless‑steel walls of the tubing, the frits at the column inlet and outlet, and the residual active groups (such as free silanols) on the silica‑based packing.

Mobile phase along cannot saturate these active sites, even if equilibrated for a long time, as these sites can be filled by specific adsorptions, but not any substances.

If an analyte has coordinating or strongly interacting functional groups (for example, cytarabine containing phosphate-related functionality, peptides, or acidic compounds with carboxylate groups), when entering a new, unsaturated flow path, some of its molecules may be strongly retained by metal ions on the frits or residual silanol groups on the packing material.

As a result, a significant fraction of the molecules in the first injection may be consumed in occupying these active sites, and therefore fewer molecules actually reach the detector, and the response is artificially low. It is only after the first injection has adsorbed enough analyte to satisfy these active sites that, from the second injection onward, the compound molecules can pass through unimpeded and responses become normal and reproducible.

The column is not the only component that can experience a cold-start effect. The autosampler also experiences it.

After completing a previous sequence or during standby, an autosampler rinses the injection needle (both inside and outside) to minimize carryover and cross-contamination. However, when the idle time is prolonged, subtle changes can occur in the liquid remaining in the needle, needle seat, and sample loop because of capillary effects and diffusion, leading to some common problems:

• Solvent dilution: Residue organic wash solvent may linger in the needle seat. During the first injection, this residual solvent can mix with the sample and effectively dilute it.
• Temperature differences and air bubbles: Small metal components in the needle and sample loop can behave differently when the autosampler is stationary compared with when it is operating continuously due to temperature differences. As a result, during the first injection, the initial movement of the syringe plunger makes the system more susceptible to forming tiny, often invisible air bubbles, reducing the actual injected volume and consequently an abnormal response.

From the second injection onward, the injection cycle has already become continuous. The flow path is thoroughly filled and flushed with sample, allowing volumetric precision to return to its normal level.

Some chromatographer may wonder, “I have equilibrated the column with the mobile phase for two hours, and the baseline is perfectly flat. Why is the first injection still unreliable?”

To answer this question, it is necessary to distinguish static equilibrium from dynamic equilibrium. Static equilibrium merely means the mobile phase flows at a steady rate, with pressure and temperature holding a straight line. Dynamic equilibrium, however, involves a cascade of physical changes from valve switching to pressure pulse, then to sample matrix alteration, and then to minor column temperature fluctuations.

When the injection valve (typically a six-port valve) switches from the load position to the inject position, the system pressure can briefly drop and then rise again. It produces a pressure pulse of a few megapascals (typically within 1 MPa), which sweeps through the column. The resulting local compression of the packing material and minute flow perturbations all imprint on the peak area of the first injection.

Only after the injection valve has switched through one or two cycles do the small changes in flow conditions, pressure, and temperature inside the column settle into the dynamic equilibrium characteristic of normal sample injection.