The Flow Cell in HPLC: A Small Component with Big Consequences
Table of contents
Introduction
In routine liquid chromatography (LC) analyses, column screening, mobile phase composition, and gradient optimization are what we often devote most of our attention to. However, the flow cell, a core component of the detector, is frequently overlooked despite its substantial influence.
In this article, we examine this inconspicuous, yet critical component.
Case Studies: How The Flow Cell Can "Ruin" Your Experiment
Case 1: Flat-topped peak dilemma
User A (a university) encountered persistent flat-topped peaks on a Sail1000 10-mL Analytical LC System due to the strong UV absorbance of the analyte, causing quantification to be impossible. Replacing the standard 10 mm path-length flow cell with a 2 mm variant resolved this issue.
(The user's method is classified and thus experiment conditions and chromatograms are not displayed here.)
Case 2: Purification bottleneck
User B (a pharmaceutical company) experienced sample overloading on an EasyPure800 Purification LC System for mid- to low-pressure purifications. Without reducing the injection amount, changing the flow cell from a 1-mm path length to a 0.3-mm one eliminated the overload issue and improved work throughput.
(The user's method is also classified and thus experiment conditions and chromatograms are not displayed here.)
Welch Materials engineers tested a standard sample on both path lengths under User B's conditions, and the results are as follows:
Case 3: Discrepancies between instruments of the same model
An online user shared an experiment where identical concentration samples produced markedly different detector responses on two instruments of the same model; troubleshooting revealed the two instruments were fitted with different flow cells.
| Instrument | Peak height | Peak area | Flow cell |
| A | 942.18 | 6174.7 | Cell volume: 5 µL Path length: 6 mm |
| B | 1567.13 | 9666.86 | Cell volume: 10 µL Path length: 10 mm |
All three users’ problems traced back to the flow cell. What secrets does this small component hold?
What Is a Flow Cell, and Which of Its Parameters Matter
The flow cell is the "eye" of a UV detector, the optical window through which sample passes and is detected. The core parameters of a flow cell include:
- Path length: determines sensitivity following the Beer–Lambert law; longer path lengths yield higher sensitivity.
- Volume: directly affects extra-column band broadening. Excessive volume can cause already-separated peaks to remix within the detector, producing peak broadening and reduced column efficiency.
- Pressure rating: a flow cell capable of withstanding high pressure is essential, especially when using small-particle columns or high-viscosity mobile phases,.
- Window material: typically quartz, applicable for the UV–visible spectral range.
Flow Cell Selection
The selection of flow cells must be based on the internal diameter (I.D.) of the column. Usually, the smaller the column I.D., the smaller the flow cell volume should be. If the flow cell is too large relative to the column, a solvent accumulation effect occurs as sample moves from the column into the flow cell, producing substantial extra-column volume effects that adversely affect separation.
When selecting flow cell volume relative to the column and instrument, a practical guideline is to choose a flow cell volume less than one-tenth (1/10) of the elution volume of the earliest (dead-time) chromatographic peak.
Besides keeping volume as small as practical, prefer flow cells that are as long as possible in their optical path dimension when selecting.
How Flow Cells Affect Assays
Generally, Larger flow cells provide better detection limits (LODs), while smaller flow cells yield better peak resolution.
A larger flow cell collects greater light flux and—owing to its longer path length—produces a stronger signal at the photodetector. This increases sensitivity, and as a result improves the limit of detection.
Smaller flow cells, while receiving less light flux, have smaller internal dimensions and shorter path lengths, which reduce diffusion within the cell and as a result improve peak resolution.
According to the Beer–Lambert law:
Absorbance = − log T = log (l₀/l) = ε × C × d
The longer the optical path length, the higher the signal intensity. Since noise usually does not increase significantly with path length, the signal-to-noise ratio improves. For example, when the path length increases from 6 mm to 10 mm, noise increases by only less than 10% while signal intensity increases by about 70%.
The increase of path length often leads to the increase of the flow cell volume (in the example above, the volume increases from 5 µL to 14 µL). This increase likely leads to some peak dispersion, but in most cases does not compromise resolution in gradient separations.
Application: Maximizing Sensitivity in Aflatoxin Analysis with Well-chosen Flow Cells
To demonstrate the above conclusions, let's consider this positive case. Aflatoxin analysis traditionally requires complex pre-column or post-column derivatization to enhance fluorescence. However, in the two examples below, researchers used large-volume flow cells, foregoing laborious and hazardous pre-column derivatization and the specialized equipment required for post-column derivatization, and still achieved signal amplification.
Case 1
Conditions:
- Column: Waters Acquity BEH C18 (2.1 mm × 100 mm, 1.7 μm)
- Mobile phase:
A: methanol : acetonitrile = 50 : 50 (v/v)
B: water
A : B = 36 : 64 (v/v) - Flow rate: 0.3 mL/min
- Column temperature: 40 °C
- Injection volume: 10 mL
- Fluorescence detector: excitation 365 nm, emission 450 nm
Under identical conditions, comparison of a conventional flow cell (2 µL) and a large-volume flow cell (13 µL) for aflatoxin response shows the large-volume cell produced a substantially higher response. Sensitivities for aflatoxin B2 and G2 increased more than fivefold; more strikingly, signal intensities for aflatoxin B1 and G1 increased about twentyfold. Therefore, this assay selected a large-volume flow cell for the fluorescence detector.
It can also be seen from the chromatogram that the use of a large flow cell increases extra-column volume effects, producing some peak tailing.
Case 2
UPLC columns elute faster than HPLC columns, significantly shortening analysis time and improving separation efficiency. Table 1 shows that lowering the flow rate can increase sensitivity, but lowered flow rates increase run time and may reduce efficiency.
Therefore, in this assay, a flow rate equal to the column manufacturer's recommended flow rate (1.0 mL/min) was chosen for the HPLC column, and 0.3 mL/min was chosen for the UPLC column. The UPLC’s lower flow rate reduces mobile phase consumption and yields obvious reagent savings.
For analytes with weak absorbance, choosing a flow cell of longer path length can increase signal strength, improving detector sensitivity and yielding better results.
For analytes with strong absorbance, flow cells of shorter path length are recommended as they better avoid signal exceeding detector's linear range, allowing higher sample loading during preparative work.
(Parts of the materials and images used in this article were sourced from the internet, literature, and the Agilent user manual. They are provided solely for discussion and non-commercial use. Please evaluate and reference them accordingly.)
References
[1] HU Jiawei et. al. Determination of 4 aflatoxin in food by ultro-high performance liquid chromatography and fluorimetric detection combined with large volume flow cell. Chinese Journal of Health Laboratory Technology, 2017, 27(8): 1109-1111.
[2] WANG Junlin et. al. Determination of aflatoxin M1 in milk by ultra-performance liquid chromatography and fluorimetric detection combined with large volume flow cell. Journal of Zhejiang University (Agriculture and Life Sciences), 2013, 39(2): 191-196.