# Development of a transportable High-Performance Liquid Chromatograph with Ultraviolet detection and a method for the rapid analysis of 13 carbonyl compounds hydrazones

Audrey Grandjean, Anaïs Becker, Mathilde Mascles, Franck Amiet, Jean-Philippe Amiet, Damien Bazin, Stéphane Le Calvé

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Audrey Grandjean, Anaïs Becker, Mathilde Mascles, Franck Amiet, Jean-Philippe Amiet, et al. Development of a transportable High-Performance Liquid Chromatograph with Ultraviolet detection and a method for the rapid analysis of 13 carbonyl compounds hydrazones. Journal of Chromatography Open, 2025, 7, pp.100201. 10.1016/j.jcoa.2024.100201. hal-05388661

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# Development of a transportable High-Performance Liquid Chromatograph with Ultraviolet detection and a method for the rapid analysis of 13 carbonyl compounds hydrazones

Audrey Grandjean<sup>a,b</sup> <sup>ID</sup>, Anaïs Becker<sup>a</sup>, Mathilde Mascles<sup>b</sup> <sup>ID</sup>, Franck Amiet<sup>b</sup>,
Jean-Philippe Amiet<sup>b</sup>, Damien Bazin<sup>b</sup>, Stéphane Le Calvé<sup>a,* ID</sup>

<figure><img src="image_3.png" /></figure>

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## ARTICLE INFO

Keywords:

Carbonyl compounds
2,4-Dinitrophenylhydrazine
transportable HPLC-UV
Isocratic analysis
ISO 16000-3 standard analysis
Hydrazones
Aldehydes

## ABSTRACT

In environments with elevated air pollution, highly reactive aldehydes pose significant health concerns. This study proposes a simultaneous analysis of 13 carbonyl compounds, according to the ISO 16,000–3 reference method, adapted for a novel transportable High-Performance Liquid Chromatograph (HPLC) system. The transportable HPLC system consists of a reinforced analytical suitcase and a computer suitcase, each equipped with spring systems to fortify them against impact. The system features a straightforward isocratic pump, ensuring reliability and ease of operation, while sample delivery is managed by a piston pump coupled to a multiplexing valve. Derived carbonyl compounds with 2,4-dinitrophenylhydrazine were separated in isocratic mode using water and acetonitrile with a reverse-phase column in less than 20 min. Out of the 13 hydrazones, only 2-butanone-2,4-DNPH (BO-DNPH) and butanal-2,4-DNPH (BA-DNPH) were fully coeluted, and separation of the other critical pairs containing 3 and 4 carbons was achieved. The method was applied to the transportable HPLC equipped with a UV or light-emitting diode (LED) detector. These results were compared with the ones obtained on two benchtop laboratory HPLCs equipped with a diode array detector (DAD). The limit of detection (LOD) for the hydrazones of the carbonyl compounds ranged from 0.12 to 0.38 mg L⁻¹ with the UV detector and 0.45 to 1.04 mg L⁻¹ with the LED detector. Precision was determined from consecutive injections of the analytes to yield RSD<11.5% (UV) and RSD<14.1% (LED). Performances were close to that of the laboratory HPLCs, either in sensitivity, resolution, or repeatability with overall more accurate and robust results with the UV than with the LED detector. Despite the LED system not fully meeting the standard method requirements with line-arisities correlation coefficients lower than 0.999, both systems and methods were validated for the isocratic analysis of hydrazones for aldehydes and ketones quantification. An additional comparison was made with the literature regarding both the isocratic method developed and the transportable system.

## 1. Introduction

Aldehydes and ketones are naturally present in fruits and other foods, in residues in the soil as decomposition products and can also be formed via the reaction of terpenes emitted by foliage with ozone and OH or NO₃ radicals in the atmosphere [1,2]. Combustion processes induce the formation of a wide range of molecules, including aldehydes. Therefore, traffic is one of the main contributors to ambient air aldehyde concentration. In confined spaces, sources include cooking activities, fireplace use, or smoking. Other main sources include furniture or

construction materials such as plywood, paints, carpets, or insulating foam. Terpenes are widely used as fragrances and flavours in consumer products and can also act as secondary sources for aldehydes emission indoors [3].

In professional settings, individuals working with aldehydes as raw materials can be exposed to higher concentrations than the general public. Formaldehyde, widely used as a biocide, finds applications in the health, food, and food-processing industries, hospital and funeral services [4,5]. Moreover, it serves as an intermediate in the chemical, pharmaceutical, and plastic industries. Other aldehydes and ketones like

<footnote><sup>a</sup> ICPEES – CNRS/University of Strasbourg, 25 rue Becquerel, Strasbourg, France</footnote>

<footnote><sup>b</sup> Chromatotec, 15 Rue d'Artiguelongue, Saint-Antoine, France</footnote>

<footnote>* Corresponding author</footnote>

<footnote>E-mail address: slecalve@unistra.fr (S.L. Calvé).</footnote>
acetone, acetaldehyde and butanone serve as solvents and chemical
intermediates in various manufacturing processes [5].

Formaldehyde, encountered at the highest concentrations compared to other aldehydes, has been classified as carcinogenic (Group 1) by the International Agency for Research on Cancer (IARC) in 2004. Acetaldehyde and acrolein may also be a human carcinogen, albeit with a lower degree of certainty. Acetaldehyde and crotonaldehyde are suspected to be mutagenic [6]. Diseases such as respiratory allergies, neurogenerative disease, contact dermatitis or liver diseases have been associated with the accumulation of aldehydes [7].

The standard procedure for formaldehyde monitoring in the indoor air or test chambers consists of sampling air through a 2,4-dinitrophe-nylhydrazine (DNPH) tube, followed by its elution with acetonitrile and injection into an HPLC with UV (or Diode Array) detection at 360 nm [8]. In the following paragraphs, concentrations will be expressed in hydrazone derivative in the liquid phase. We will consider a typical case where the sampling volume is fixed at 0.06 m³ (1 L min⁻¹ for 1 hour) and eluted with 4 mL of acetonitrile. A table of the equivalence of aldehyde concentration in the air to their corresponding hydrazone in the liquid phase for different sampling and elution volumes is provided in the Supporting Information (Table SI-1, Equation SI-1).

Mainly driven by anthropic sources, the resulting concentrations of formaldehyde in rural outdoor, urban outdoor and indoor environments usually vary in the ranges 0.5–5, 5–20 and 10–50 µg m⁻³ (or, in the equivalent concentration of hydrazone when collected as described above: 0.05–0.5, 0.5–2 and 1–5 mg L⁻¹), respectively. The second most abundant carbonyl compounds detected in the air are acetaldehyde and acetone, with concentrations typically between 5 and 15 µg m⁻³ (0.3–1 mg L⁻¹ of the corresponding hydrazone in liquid phase) in indoor air and 1–3 µg m⁻³ (0.06–0.2 mg L⁻¹ hydrazone) in outdoor air. Other detected carbonyl compounds in indoor air include acrolein (< 2 µg m⁻³, < 0.1 mg L⁻¹ hydrazone), propionaldehyde (0–15 µg m⁻³, 0–1 mg L⁻¹ hydra-zone), butanal (indoor < 3 µg m⁻³, < 15 mg L⁻¹ hydrazone), benzalde-hyde (0–8 µg m⁻³, 0–0.3 mg L⁻¹ hydrazone), and hexanal (5–20 µg m⁻³, 0.2–0.8 mg L⁻¹ hydrazone) [9,10]. Outdoors, they are usually present at concentrations below 1 µg m⁻³ (0.05 mg L⁻¹ hydrazone) but the con-centrations can be tenfold more in polluted urban areas [11].

Guidelines exposure values have been fixed by the World Health
Organisation for formaldehyde at 100 µg m⁻³ (10.5 mg L⁻¹ corre-
sponding hydrazone in the liquid phase) for short-term exposure (30
min) to prevent sensory irritation in the general population [2].
Regarding long-term exposure, French authorities recommend a limit of
30 µg m⁻³ (3.1 mg L⁻¹ hydrazone) in establishments receiving public
[12]. Occupational exposure is regulated for formaldehyde and other
carbonyl compounds for a period of 8 h: 0.37 mg m⁻³ (39 mg L⁻¹
hydrazone) for formaldehyde (FA), 180 mg m⁻³ (13 700 mg L⁻¹ hydra-
zone) for acetaldehyde, 0.05 mg m⁻³ (3.1 mg L⁻¹ hydrazone) for acro-
lein, 1210 mg m⁻³ (74 mg L⁻¹ hydrazone) for acetone, 600 mg m⁻³ (31
500 mg L⁻¹ hydrazone) for butanone and 175 mg m⁻³ (8100 mg L⁻¹
hydrazone) for valeraldehyde [13]. It is interesting to note that acrolein
has the lowest value for occupational exposure, suggesting that this
pollutant is highly hazardous to health.

Due to its high toxicity and difficulty to be detected, efforts were put into formaldehyde-specific monitoring techniques. Differential optical absorption spectroscopy (DOAS), cavity ring-down spectroscopy (CRDS), Fourier transform infrared spectroscopy (FTIR) coupled with photoacoustic spectroscopy or Hantzsch fluorimetry have been demonstrated to accurately quantify formaldehyde at low concentrations [4,14,15]. Portable techniques for airborne formaldehyde quantification such as colorimetry can be used for concentrations in the range of tens µg m⁻³ to mg m⁻³ (mg L⁻¹ to hundreds of mg L⁻¹ of corresponding hydrazone) but often suffer from interference with other aldehydes [16].

To simultaneously analyse a wide range of aldehydes and ketones, separating techniques such as gas chromatography or liquid chromatography coupled to a detector are commonly used. To enhance detection properties such as selectivity and sensitivity and give stability to the

short-chain carbonyl compounds, these methods involve a derivatisation step before analysis. Carbonyl compounds react with hydrazine to form hydrazones [10] or with amines to form oximes [17]. The standard methods TO-11 A [18] and ISO 16,000–3 [8] involving DNPH derivatisation and HPLC/UV analysis are the most widely used technique for formaldehyde and other carbonyl compounds monitoring in the air [19–24]. The use of DNPH for the analysis of formaldehyde in other media has also been reported many times, such as for food [25] or rainwater [26]. Other systems use a completely different technique from the standardised method for the targeted compounds. For instance, Hantzsch reaction and fluorimetry detection have been used instead of DNPH derivation and HPLC-UV analysis for specific formaldehyde analysis [15].

The standard procedure involving DNPH sampling and HPLC/UV analysis, while proven sensitive and reliable, suffers from a few drawbacks. First, it is an indirect method that requires a trained operator. This work constitutes the first step to simplify and automatise on-site use of the method. Secondly, both before and after sampling, DNPH tubes must be stored in the dark and at 4 °C to avoid degradation. Manipulation of the tube after sampling and before the analysis is crucial, as careless storage can directly impact the results. Minimising the time between sampling and analysis by bringing a transportable HPLC onto the field can reduce the likelihood of such events. In this work, direct analysis after on-field sampling was made possible, hence removing the issue of post-sampling storage.

In the past decade, efforts have been made to bring laboratory analytical systems directly into the field through miniaturisation of instruments for either targeted detection, such as formaldehyde-specific techniques [15], or for non-targeted characterisation [27]. Some of these modified systems are miniaturised to the detriment of other parameters such as range of application. For example, light-emitting diode (LED) detectors for miniaturised HPLC are lightweight but emit at a single wavelength, thus limiting their use to the selected target analytes [28,29]. In addition, LED light is not necessarily perfectly monochromatic, which can lead, for example, to undesirable interferences.

This work involves both the development of a portable analytical device and the development of an analytical method. Indeed, this work had a double objective: i) to develop a robust, transportable HPLC for future on-site analysis of carbonyl compounds in air sampled on DNPH tubes; ii) to develop and validate an ISO 16,000-3-compliant method for quantifying derivatives of 13 carbonyl compounds (including formaldehyde) after reaction with DNPH. To our best knowledge, the developed method was the only isocratic method in the literature achieving the separation of 11 of the 13 hydrazones with UV detection in less than 20 min, and with a detection limit below 1 mg L⁻¹ for formaldehyde-2,4-dinitrophenylhydrazone (FA-DNPH). The HPLC system for which the method was developed was designed to keep the robustness, sensitivity and potential of a classic laboratory system while being transportable for reliable and fast on-site analysis. The system with either a UV detector with a fixed wavelength or an LED detector was compared to two other bench laboratory HPLC or UHPLC systems.

## 2. Material and method

### 2.1. Chemicals

Carbonyl-DNPH samples for analysis were prepared from two standard solutions of 13 hydrazones. "Standard 1" contained hydrazones at an equal concentration of 100 µg mL⁻¹ (Mix 13 – 100 µg mL⁻¹, NEO-CHEMA GmbH, Bodenheim, Germany). "Standard 2" contained hydrazones at various concentrations corresponding to equal concentrations of their carbonyl equivalent at 30 µg mL⁻¹ (CARB Method 1004 DNPH Mix 2 at 30 µg mL⁻¹, Merck, Massachussets, USA). Each of them was further diluted in acetonitrile (gradient grade ≥99.9%, Honeywell, North Carolina, USA). The standards were stored at 4 °C in amber, airtight bottles after opening. They were used for a maximum of two
months after opening.

## 2.2. Apparatus

Four commercialised HPLC systems were used in this study (Figure SI-1, Figure SI-2, Table SI-2). The two bench laboratory systems used as a reference in terms of analytical performances were: an Agilent 1100 series (HPLC A) and a Shimadzu UHPLC Nexera X2/XR/AR (HPLC B). They were equipped with a degasser and a diode array detector. The Agilent 1100 series was a twenty-year-old technology, while the other was much more recent and was purchased in 2019.

The Chromatotec HPLCs (HPLC C-UV and HPLC C-LED) were equipped with an AZURA® Pump P 2.1S compact HPLC pump (Knauer, Berlin, Germany), a 10-Way valve and a Cavro Pulssar PBC piston pump (Tecan, Männedorf, Switzerland) for sample delivery, a 6-way injection valve (Valco, VICI, Houston, USA) with a 5 µL PEEK sample loop, and a detector. The detector was the only difference between the two HPLC chains C-UV and C-LED: HPLC C-UV used a variable single wavelength UV detector (AZURA UV Detector UVD 2.1S) set at 360 nm. In contrast, HPLC C-LED used an LED emitting at 365 nm and a photodiode sensor. Both systems were operated from Vistachrom (v1.6) software. HPLC A and B are lab bench modules with a separate computer. HPLC C comprises a reinforced analytical suitcase designed to absorb transportation impacts thanks to incorporated springs. The analytical components alone weigh 11.7 kg for a total of 23 kg once included in the reinforced suitcase. A separate computer suitcase (19 kg) integrating all the electronic parts was connected to the analytical suitcase via 3 cables ensuring a quick installation (less than 1 hour). Both suitcases respect most airlines' luggage weight restrictions.

HPLC A and B (Figure SI-3) have typical configurations of conventional laboratory HPLC. HPLC C-UV and HPLC C-LED, present an overall similar configuration. Hence, only HPLC C-UV was schemed in Fig. 1.

Prior to HPLC C usage, the mobile phase is prepared by mixing 750 mL of acetonitrile with 250 mL of water. It is partially degassed using an ultrasonic bath. A single pump pumps the mobile phase through the loop, the column, and the detector to a waste bottle (see Fig. 1). Sample delivery is managed by a 10-way valve connected to the sample vials and a piston pump. 8 ports of the 10-way valve can be used for sample analysis while the 2 other ports are used for sample waste and rinsing of the multiplexing system. At the start of a run, the 6-way valve is switched to put the sample loop in line with the sample delivery system.

A piston pump draws the sample through the valves until the loop is full. The 6-way valve is switched back to inject the analytes into the column for separation and detection with the UV detector. Filters were placed after the pump and before the column for additional protection of the column. A backpressure regulator was added after detection to minimise chances of bubbles formation in the detection cell. The LED detector allowed a single wavelength use determined by the 365 nm emitting diode.

## 2.3. Method parameters

A preliminary study was conducted with several HPLC columns and isocratic conditions, the ProntoSIL™ 120–3 C18 AQ column (25 cm × 3 mm × 3 µm) exhibited the best separation.

Chromatographic method parameters are summarised in Table 1 (Detection parameters in Table SI-3).

The detection wavelength was set at 360 nm for both UV and DAD detectors as stipulated in the standard procedure [8]. The LED was chosen because of its miniaturisation potential due to the absence of complex and cumbersome optics. This type of LED was only available at 365 nm with a spectral bandwidth of 15 nm. The absorption peak being broad, 365 nm is still in the maximum absorption region of the hydro-zones. HPLC A and B were equipped with “analytical/precision” (as opposed to preparative) flow cells with a 10-mm optical path length. HPLC C-UV and C-LED were equipped with an identical flow cell with a 3 mm path length. The acquisition frequency was fixed at 5 Hz for HPLC A and C, and a similar value for HPLC B, giving 5 points per second, which was sufficient to define an HPLC chromatographic peak of 10 to 40 s wide.

<table><caption>**Table 1**
Chromatographic parameters for the method 1 (0.4 mL min⁻¹).</caption><thead><tr><th>Column</th><th>ProntoSIL™ 120–3 C18 AQ 25 cm × 3 mm, 3 µm</th></tr></thead><tbody><tr><td>Mobile phase</td><td>Acetonitrile/H<sub>2</sub>O 75:25</td></tr><tr><td>Flow rate</td><td>0.4 mL min<sup>-1</sup></td></tr><tr><td>Injection volume</td><td>5 µL</td></tr><tr><td>Oven temperature</td><td>33 °C</td></tr><tr><td>Sample</td><td>13 Carbonyl-DNPH standards</td></tr></tbody></table>

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Detailed scheme of HPLC C-UV.</figcaption></figure>
### 3. Results and discussion

This section first presents the optimisation of chromatographic conditions, and the validation of the method where the linearity, the detection limits, the repeatability, and the determination of chromatographic resolution were investigated. Then, a comparison of the analytical performances of the method applied to the transportable HPLCs to those obtained with conventional HPLCs was made. Finally, the comparison extended to other isocratic methods found in the literature, and the non-chromatographic characteristics of the transportable HPLCs were considered alongside other transportable HPLCs.

#### 3.1. Optimisation of chromatographic conditions

Six C18 columns with various lengths, internal diameter and particle size were first tested on HPLC A. For each column, the isocratic condition of elution was optimised. The ProntoSIL™ 120–3 C18 AQ showed better performance in terms of separation than its other C18 counterparts (see Figure SI-4) and was thus selected for the next experiments. The improved performance for the separation of hydrazone compounds observed with the ProntoSIL™ 120–3 C18 AQ column might be due to the hydrophilic end-capping, causing enhanced hydrogen bonding and silanol activity [30], and finer granulometry (3 µm instead of 5 µm for

the other columns).

Chromatograms in the optimised conditions (Table 1) with all four HPLCs are plotted in Fig. 2 for the lowest injected concentration of standard solution of hydrazones. 12 peaks are present for 13 hydrazone compounds. All compounds are separated except for BO-DNPH and BA-DNPH, which are fully coeluted. These two compounds could be partially separated when using a higher proportion of water in the mobile phase. However, it significantly increased the retention time and consequently reduced the sensitivity of the last 4 compounds. A compromise in water/acetonitrile proportion was made to maintain satisfactory HA-DNPH detection at the expense of the BO and BA peak separation.

In Fig. 2, the injection peak is visible at about 2.5 min and FA-DNPH eluted at about 5 min. It is followed by hydrazones with increasing chain lengths and/or with a higher degree of saturation. The last compound, HA-DNPH, was eluted at 14.5 min for HPLC A, 17.1 min for HPLC B, 14.4 min for HPLC C-UV and 14.9 min for HPLC C-LED. The difference in retention times between HPLC B and the other 3 HPLCs is due to the volume between the outlet of the column and the inlet of the detector. The tubing length of HPLC B was twice as long compared to the other HPLCs, favouring dispersion of the analytes and larger peak width.

<table><thead><tr><th rowspan="2">Peak n°</th><th rowspan="2">Compound</th><th colspan="2">Concentration (mg L<sup>-1</sup>)</th><th colspan="4">Retention time (min)</th></tr><tr><th>HPLC A; B</th><th>HPLC C</th><th>HPLC A</th><th>HPLC B</th><th>HPLC C-UV</th><th>HPLC C-LED</th></tr></thead><tbody><tr><td>1</td><td>FA-DNPH</td><td>1.0</td><td>7.0</td><td>5.0</td><td>5.6</td><td>4.7</td><td>5.0</td></tr><tr><td>2</td><td>AA-DNPH</td><td>1.0</td><td>5.1</td><td>5.7</td><td>6.5</td><td>5.5</td><td>5.7</td></tr><tr><td>3</td><td>AC-DNPH</td><td>1.0</td><td>4.1</td><td>6.5</td><td>7.5</td><td>6.3</td><td>6.6</td></tr><tr><td>4</td><td>AO-DNPH</td><td>1.0</td><td>4.2</td><td>6.8</td><td>7.7</td><td>6.5</td><td>6.8</td></tr><tr><td>5</td><td>PA-DNPH</td><td>1.0</td><td>4.1</td><td>7.1</td><td>8.0</td><td>6.8</td><td>7.1</td></tr><tr><td>6</td><td>CA-DNPH</td><td>1.0</td><td>3.6</td><td>7.9</td><td>9.1</td><td>7.7</td><td>8.1</td></tr><tr><td>7</td><td>MC-DNPH</td><td>1.0</td><td>3.6</td><td>8.3</td><td>9.6</td><td>8.1</td><td>8.5</td></tr><tr><td>8</td><td>BA-DNPH + BO-DNPH</td><td>1.0</td><td>3.5</td><td>8.7</td><td>10.0</td><td>8.4</td><td>8.8</td></tr><tr><td>9</td><td>BZ-DNPH</td><td>1.0</td><td>3.5</td><td>9.3</td><td>10.8</td><td>9.1</td><td>9.5</td></tr><tr><td>10</td><td>VA-DNPH</td><td>1.0</td><td>2.7</td><td>11.0</td><td>12.9</td><td>10.9</td><td>11.3</td></tr><tr><td>11</td><td>TA-DNPH</td><td>1.0</td><td>3.1</td><td>11.9</td><td>14.0</td><td>11.8</td><td>12.3</td></tr><tr><td>12</td><td>HA-DNPH</td><td>1.0</td><td>2.5</td><td>14.5</td><td>17.1</td><td>14.4</td><td>14.9</td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>Fig. 2. (a) Chromatograms of a standard solution of 13 carbonyl-DNPH diluted to 1 mg L⁻¹ for HPLC A and B, and diluted to hydrazones concentrations ranging between 2.5 to 7 mg L⁻¹ for HPLC C-UV and HPLC C-LED and their (b) corresponding peak identification, concentration, and retention time.</figcaption></figure>
## 3.2. Characterization of the method

### 3.2.1. Linearity

Calibration curves for the 13 hydrazones with the developed method on HPLC A, B, C-UV, and C-LED were plotted. The individual curves for each hydrazone with each HPLC can be found in Figures SI-5-SI-8.

Slope values and regression coefficients are listed in Table SI-4. HPLC A and HPLC B exhibit excellent linearities with coefficients above 0.9999 for most of the compounds. HPLC C-UV shows regression coefficients above 0.999, while HPLC C-LED coefficient reaches R² values above 0.99 for all hydrazones. According to the ISO 16,000–3 standard method, an appropriate linear response is obtained when the regression coefficient is higher than 0.999. In this series of experiments, HPLC A and C-UV fulfil this requirement. HPLC B also fulfils it for all compounds except FA-DNPH (R² = 0.9977). HPLC C-LED fulfils it for VA-DNPH, TA-DNPH and HA-DNPH but not for the rest of the compounds. Therefore, LED detection must be improved to satisfy the standard linearity requirement.

Slopes between HPLC cannot be compared directly since the response signal is given in arbitrary units. However, tendencies between molecules for the same HPLC can be highlighted. MC-DNPH has the highest slope coefficient (77.7 for HPLC A), and TA-DNPH and HA-DNPH have the lowest slope coefficient (56.5 and 58.2 for HPLC A). The trend is similar for the other HPLCs, and all the slopes vary within 10.6%, 10.0%, 12.3% and 21.6% (RSD) for HPLC A, B, C-UV, and C-LED, respectively. At 360 nm, the highest contributing group for absorption is the DNPH chromophore, which is a common part of all the analysed molecules. Therefore, their absorption capacity is close, and the slope values are similar.

### 3.2.2. Detection limits

The limits of detection of the method for each hydrazone and each HPLC were calculated in three different ways (Equations SI-2, SI-3 and SI-4). The most common one for the LOD determination by HPLC analysis is the height method, represented for our data in Fig. 3. They

were determined from the lowest injected concentration used for the linearities. The limits of detection vary depending on the hydrazone in the range of 0.22 to 0.60 mg L⁻¹ for HPLC A, 0.02 to 0.05 mg L⁻¹ for HPLC B, 0.12 to 0.38 mg L⁻¹ for HPLC C-UV and 0.45 to 1.04 mg L⁻¹ for HPLC C-LED. As expected, the limit of detection of HPLC B is significantly lower than that of its counterparts since this is a very recent bench laboratory HPLC. In addition, its flow cell has a long optical path length (10 mm) for a volume of 1 µL, while HPLC C-UV and C-LED have a 3-mm optical path length for a volume of 2 µL. Longer optical path length and smaller volume allow increased sensitivity while keeping noise low. HPLC B is a UHPLC system with a detector using fibre optics to achieve such low volume and long path length. HPLC A also has a 10-mm optical path length, but for a volume of 13 µL, and belongs to the older generation of HPLC. The sensitivity achieved for each HPLC is therefore consistent with its characteristics.

Two other methods were used to calculate the limits of detection, namely the US EPA and EN 14,662–3 methods, symbolised by crosses and triangles in Fig. 4 while the height method is symbolised by dots. For the US EPA and EN 14,662–3 methods, the same concentration of analyte was consecutively injected 10 times into the four HPLCs. For this, a low concentration above the LOD already determined by the height method was used. Every method gave consistent results, and usually, higher LOD was observed with the height method compared to the other two. These US EPA and EN 14,662–3 methods depend on the standard deviation of the concentration obtained from 10 injections, hence on the repeatability.

The first step of the method ISO 16,000–3 for the analysis of aldehydes in the air is air sampling through a DNPH tube and subsequent elution of the tube with a few millilitres of acetonitrile. Since this step was not subject to any improvement in this study, it was not included in the development. However, to give an idea of the practical application of the HPLC method, the theoretical equivalent detection limits of the carbonyl compounds in the gas phase are available in Table SI-5.

<figure><img src="image_6.png" /><figcaption>Fig. 3. (a) Limit of detection (LOD) based on the height method for the 13 carbonyl-DNPH analysed on the 4 HPLC systems using an isocratic elution of ACN: Water 75:25, an oven temperature of 33 °C and an injection volume of 5 µL. The LOD was calculated from the lowest value of the linearity plots (b) Injected concentration of hydrazones in mg L⁻¹.</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Fig. 4. Detection limits of 13 carbonyl-DNPH compounds with the height, the US EPA and the EN 14,662–3 methods for HPLC A, B, C-UV and C-LED.</figcaption></figure>

### 3.2.3. Repeatability

To evaluate the repeatability, a low concentration of the hydrazone standard solution above LOD levels was injected 10 consecutive times into each HPLC. So that all hydrazone concentrations of this standard mixture were above the LOD determined in Figs. 3 and 4 (except for TA-DNPH and HA-DNPH on HPLC A with the height method only). The

relative standard deviation of the areas obtained for each compound is shown in Fig. 5 (data in Table SI-6). RSDs of HPLC B peaks were all below 3.6%, demonstrating its excellent repeatability. RSDs of peak areas obtained with HPLC A were between 5.6 and 13.8%, with a tendency to increase with retention time. With HPLC C-UV, RSD values remained below 10%, except for MC-DNPH (11.5%). The same

<figure><img src="image_8.png" /><figcaption>Fig. 5. (a) The relative standard deviation of peak area for 10 consecutive injections of the standard mix with varied concentrations (b) injected concentrations of the hydrazones in mg L⁻¹.</figcaption></figure>
behaviour was observed for HPLC C-LED except for HA-DNPH (14.1%). According to the standard method, a response within 10% for analytes at 1 mg L⁻¹ or more and 20% for analytes at a concentration near 0.5 mg L⁻¹ is expected. Repeated injections showed that the two transportable configurations of HPLC comply with the requirement with all compounds except MC-DNPH for HPLC C-UV and HA-DNPH for HPLC C-LED. The recent bench laboratory HPLC B and more ancient HPLC A also comply with the lower than 20% RSD requirement.

### 3.2.4. Resolution

Resolution values (R) based on the chromatograms in Fig. 2 were calculated with the Tangent method (Equation SI-5) and reported in Table 2. As a visual guide, values of 0.5, 1 and 1.5 were also represented in green, orange and red colours. Resolution values were always above 0.5 for all compounds and all HPLCs. Peaks (1) (2) (9) (10) (11) and (12) were always above 2, i.e., fully separated. HPLC A, initially used for method development, demonstrates the best peak resolutions. R values for C₃ peaks (corresponding to carbonyl compounds with a chain length of 3 carbon atoms), peaks (3) (4) (5), were 1.49, 1.49 and 1.61. R values for C₄ peaks (corresponding to carbonyl compounds with a chain length of 4 carbon atoms), peaks (6) (7) (8) (9) were 2.09, 1.81 and 1.81. HPLC C-UV follows closely with peaks (3–5) at 1.20, 1.20, and 1.46 and peaks (6–8) at 1.66, 1.39 and 1.39. However, C₃ peak resolutions (3–5) were mostly below 1 for HPLC B (0.69, 0.69, 0.95) and for HPLC C-LED (0.87, 0.87, 1.06), while C₄ peaks (6–8) were between 1 and 1.5: 1.27, 1.09, 1.09 for HPLC B and 1.33, 1.33, 1.14 for HPLC C-LED. Due to the configuration of HPLC B, the tubing linking the column to the detector is rather long. It induces greater dispersion than for the other HPLCs, hence poorer resolution. The tubing length of HPLC C-UV is reduced because the system is compact compared to benchtop HPLCs. It is also the case for HPLC C-LED but resolution values for C₃ and C₄ clusters were like that of HPLC B. The detection cell of HPLC-LED being identical to HPLC C-UV, resolution results reflect the detector performance, possibly the information processing speed.

### 3.3. Comparison with the literature

In the following part, we present a comparison with the literature on two aspects of our work. First, a focus is put on isocratic HPLC methods for the analysis of carbonyl compounds. Then, we will bring attention to the portability of the HPLC.

#### 3.3.1. HPLC method

The DNPH/HPLC-UV method has been the one chosen for standard analysis of formaldehyde and other carbonyl compounds. It allows simultaneous detection of several saturated, unsaturated, dicarboxylic and aromatic aldehydes and ketones [31]. The method is robust and reliable, often employing gradient analysis [25] for best peak separation. The C₃ hydrazones (of acrolein, acetone, propionaldehyde) and C₄ hydrazones (of crotonaldehyde, methacrolein, butanone, butanal) are the most challenging to separate. Few isocratic methods are developed for laboratory equipment because it is of little interest. But to diminish the cost as well as reduce its weight for better transportability, HPLC C was equipped with a single pump, able to perform isocratic elution only. The isocratic method was developed to be used on this HPLC. Other isocratic methods found in the literature for the DNPH/ HPLC-UV analysis of aldehydes and ketones are summarised in Table 3 where the HPLC instruments, the HPLC column, the eluant, the elution flow rate, the targeted analytes, the run duration, and the LOD for FA-DNPH are listed. All the HPLC systems reported were equipped with either an isocratic of quaternary pump, a C18 column and a UV–Vis or diode array detection system fixed at 360 or 365 nm. Chromatograms and validation parameters were not systematically reported, which may limit the points of comparison and the discussion that follows.

For reasons of lower toxicity, the use of acetonitrile is preferred over methanol. Most of the studies used acetonitrile as visible in Table 3 except four reported works [23,32–34]. The ratio varied from 50:50 [35] to 77:33 [19] for ACN/H₂O and 75:25 [34] to 80:20 (with 0.5% acetic acid) [33] for MeOH/H₂O. The optimal balance between solvents of different elution strengths strongly depends on the column used.

Columns reported differed in dimensions: from 110 to 250 mm in length, 3 to 4.7 mm in internal diameter and a granulometry of 2.7 to 5 µm. The column used in this work's method was amongst the longest (250 mm), with the shortest internal diameter (3 mm) and finest granulometry (3 µm). Most methods found in the literature used shorter columns with greater granulometry.

For example, Grosjean and Grosjean (1995) analysed forty-two carbonyls including aliphatic aldehydes, aliphatic ketones, aromatic aldehydes, hydroxycarbonyl, oxo-acids and dicarbonyls using a Partisphere (5 µm, 110 × 4.7 mm) and an Axxiom ODS (5 µm, 150 × 4.7 mm) [31]. They used both UV and mass spectrometry (MS) detection. Although their resolution values were not provided, we can deduce from the extremely close retention times reported (a few or less than a few

<caption>**Table 2**</caption>

Resolution values (USP) calculated from chromatograms of each HPLC system for the concentration analysed carbonyl-DNPH standard (1 mg L⁻¹ for HPLC A and B, and from 2.5 to 7 mg L⁻¹ for HPLC C-UV and HPLC C-LED). Cases are coloured in red, orange, and green when R is above 0.5, 1 and 1.5, respectively.

<table><thead><tr><th>Peak</th><th>Compound</th><th>HPLC A</th><th>HPLC B</th><th>HPLC C-UV</th><th>HPLC C-LED</th></tr></thead><tbody><tr><td>1</td><td>FA-DNPH</td><td>3.00</td><td>2.90</td><td>3.00</td><td>3.00</td></tr><tr><td>2</td><td>AA-DNPH</td><td>3.00</td><td>2.90</td><td>3.00</td><td>3.00</td></tr><tr><td>3</td><td>AC-DNPH</td><td>1.49</td><td>0.69</td><td>1.20</td><td>0.87</td></tr><tr><td>4</td><td>AO-DNPH</td><td>1.49</td><td>0.69</td><td>1.20</td><td>0.87</td></tr><tr><td>5</td><td>PA-DNPH</td><td>1.61</td><td>0.95</td><td>1.46</td><td>1.06</td></tr><tr><td>6</td><td>CA-DNPH</td><td>2.09</td><td>1.27</td><td>1.66</td><td>1.33</td></tr><tr><td>7</td><td>MC-DNPH</td><td>1.81</td><td>1.09</td><td>1.39</td><td>1.33</td></tr><tr><td>8</td><td>BA-DNPH + BO-DNPH</td><td>1.81</td><td>1.09</td><td>1.39</td><td>1.14</td></tr><tr><td>9</td><td>BZ-DNPH</td><td>2.97</td><td>2.14</td><td>2.69</td><td>2.32</td></tr><tr><td>10</td><td>VA-DNPH</td><td>3.00</td><td>2.60</td><td>3.00</td><td>2.54</td></tr><tr><td>11</td><td>TA-DNPH</td><td>3.00</td><td>2.60</td><td>3.00</td><td>2.54</td></tr><tr><td>12</td><td>HA-DNPH</td><td>3.00</td><td>3.00</td><td>3.00</td><td>3.00</td></tr></tbody></table>
<table><caption>**Table 3**

List of isocratic methods for the analysis of hydrazone found in the literature. Only methods with more than 3 target carbonyl compounds were included.</caption><thead><tr><th rowspan="2">HPLC instrument</th><th rowspan="2">Column<sup>a</sup></th><th rowspan="2">Eluant<sup>b</sup></th><th rowspan="2">Flow rate mL min<sup>-1</sup></th><th rowspan="2">Target analytes<sup>c</sup></th><th rowspan="2">Run duration min</th><th colspan="2">LOD<sub>FA-DNPH</sub></th><th rowspan="2">Reference</th></tr><tr><th>mg L<sup>-1</sup></th><th>µg</th></tr></thead><tbody><tr><td>Jasco (880-PU, 875 UV), Rheodyne (7125), HP (3396A)</td><td>Hypersil ODS (250 × 4.6 mm)</td><td>MeOH/ACN/H<sub>2</sub>O 60:33:7</td><td>1.0</td><td>FA, AA, AC, AO, PA, CA, MVK<sup>d</sup>, MC, BA, BO, BZ</td><td>&gt; 21</td><td>—</td><td>—</td><td>[23]</td></tr><tr><td rowspan="2">Hewlett-Packard (HP) model 1090/Series II</td><td>Partisphere (5 µm, 110 × 4.7 mm)<sup>d</sup></td><td>ACN/H<sub>2</sub>O 55:45</td><td>1.0</td><td>FA, AA, PA, BA, VA, HA, IA, AC, CA, MC, AO, BO, MVK<sup>e</sup>, BZ, TA + 27<sup>e,f</sup></td><td>&gt; 14</td><td>2.4<sup>g</sup></td><td>49</td><td>[31]</td></tr><tr><td>Axxiom ODS (5 µm, 150 × 4.7 mm)<sup>d</sup></td><td>ACN/H<sub>2</sub>O 54:46</td><td>1.4</td><td></td><td>&gt; 18</td><td></td><td></td><td></td></tr><tr><td>Waters LCM1</td><td>Waters C18 (4 mm, 60 Å)</td><td>ACN/H<sub>2</sub>O 65:35</td><td>—</td><td>FA, AA, AC, PA, CA, BA, BZ</td><td>—</td><td>—</td><td>—</td><td>[26]</td></tr><tr><td>HPLC-UV (Kontron)</td><td>Supelcosil™ (250 × 4.6 mm)</td><td>ACN/H<sub>2</sub>O 60:40</td><td>1.5</td><td>FA, AA, PA, BZ, HA</td><td>&gt; 19</td><td>0.01<sup>g</sup></td><td>0.17</td><td>[21,22]</td></tr><tr><td>Hewlett-Packard 1100 series</td><td>Inertsil ODS (5 µm, 250 mm × 4.6 mm)</td><td>ACN/H<sub>2</sub>O 65:35</td><td>1.0</td><td>FA, AA, PA, BA, VA, HA</td><td>30</td><td>—</td><td>—</td><td>[40]</td></tr><tr><td>Agilent 1100 Series</td><td>Nucleosil (150 × 4.6 mm)</td><td>MeOH/H<sub>2</sub>O 75:25</td><td>1.0</td><td>FA, AA, PA, BA, VA, HA + 4<sup>e,f</sup></td><td>58</td><td>—</td><td>—</td><td>[34]</td></tr><tr><td>Hitachi (L-2130, I-2400)</td><td>Inertsil ODS-80A (5 µm, 150 × 4.6 mm)</td><td>ACN/H<sub>2</sub>O 50:50</td><td>1.0</td><td>FA, AA, AO, AC, PA, CA</td><td>&gt; 21</td><td>—</td><td>—</td><td>[37]</td></tr><tr><td>Intralab 5100</td><td>C18 analytical column</td><td>MeOH/ACN/H<sub>2</sub>O 74.5:0.5:25</td><td>1.0</td><td>FA, AA, AC, PA, AO, BA</td><td>—</td><td>0.24<sup>g</sup></td><td>—</td><td>[32]</td></tr><tr><td>Perkin-Elmer Series 200 isocratic pump and detector</td><td>Novapak C18 (4 µm, 150 × 3.9 mm)</td><td>ACN/H<sub>2</sub>O 50:50</td><td>1.5</td><td>FA, AA, AC, AO, PA, BA, BZ</td><td>—</td><td>—</td><td>—</td><td>[35]</td></tr><tr><td>Agilent 1200 Series</td><td>Eclipse XDB C18 (5 µm, 150 × 4.6 mm)</td><td>ACN/H<sub>2</sub>O 60:40</td><td>1.0</td><td>FA, AA, AC, AO, PA, CA, BO, MC, BA, BZ, VA, TA, HA + 1<sup>e,f</sup></td><td>—</td><td>—</td><td>—</td><td>[36]</td></tr><tr><td>Shimadzu (LC-5A, SPD-2A), Rheodyne (7010), HP (3395)</td><td>(5 µm, 250 × 4.6 mm)</td><td>MeOH/H<sub>2</sub>O 80:20 (0.5% acetic acid)</td><td>1.0</td><td>FA, AO, BO, BZ + 3<sup>e,f</sup></td><td>—</td><td>—</td><td>—</td><td>[33]</td></tr><tr><td>Shimadzu (LC-20AD, SIL-20AC, SPD M20A DAD<sup>h</sup>)</td><td>Ascentis Express RP- Amide (2.7 µm, 150 × 4.6 mm)</td><td>ACN/H<sub>2</sub>O 60:40</td><td>0.6</td><td>FA, AA, AO, AC, PA</td><td>&gt; 20</td><td>—</td><td>—</td><td>[38]</td></tr><tr><td>PerkinElmer Series 200</td><td>Brownlee choice C18 (5 µm, 150 × 4.6 mm)</td><td>ACN/H<sub>2</sub>O 70:30</td><td>1.5</td><td>FA, AA, PA, BZ, HA + ?<sup>f</sup></td><td>—</td><td>—</td><td>—</td><td>[20]</td></tr><tr><td>Shimadzu HPLC (LC-20AD, UV)</td><td>AichromBond-AQ C18 (5 µm, 150 × 4.6 mm)</td><td>ACN/H<sub>2</sub>O 60:40</td><td>1.2</td><td>AA, AO, PA, BA, BO, BZ, VA, HA</td><td>—</td><td>—</td><td>—</td><td>[39]</td></tr><tr><td>Lab Alliance Series 1500</td><td>Hichrom (5 µm, 250 × 4.6 mm)</td><td>ACN/H<sub>2</sub>O 70:30</td><td>1.5</td><td>FA, AA, PA, BA, IA<sup>e</sup>, VA</td><td>15</td><td>0.01<sup>g</sup></td><td>0.19</td><td>[41]</td></tr><tr><td>Agilent 1260</td><td>—</td><td>—</td><td>—</td><td>FA, AA, AO, AC, PA, CA, BA, BZ, IA<sup>e</sup>, VA, TA, HA + 1<sup>e,f</sup></td><td>—</td><td>—</td><td>—</td><td>[24]</td></tr><tr><td>Jasco (2089PU, 2010MD)</td><td>Unisol C18 (5 µm, 4.6 × 250 mm)</td><td>ACN/H<sub>2</sub>O 77:33</td><td>1.0</td><td>FA, AA, AO, AC, PA, CA, MC, BA, BO, BZ, VA, TA, HA</td><td>—</td><td>0.03–0.07<sup>g</sup></td><td>0.7–1.4<sup>g</sup></td><td>[19]</td></tr><tr><td>Agilent (A)</td><td>Prontosil™ C<sub>18</sub>-AQ (3 µm, 250 × 3 mm)<sup>g</sup></td><td>ACN/H<sub>2</sub>O 70:30</td><td>0.4</td><td>FA, AA, AO, AC, PA, CA, MC, BA, BO, BZ, VA, TA, HA</td><td>16</td><td>0.22</td><td>1.09</td><td rowspan="3">This work</td></tr><tr><td>Shimadzu (B)</td><td></td><td></td><td></td><td></td><td>21</td><td>0.02</td><td>0.12</td></tr><tr><td>Chromatotec-UV (C-UV)</td><td></td><td></td><td></td><td></td><td>17</td><td>0.12</td><td>0.60</td></tr><tr><td>Chromatotec-LED (C-LED)</td><td></td><td></td><td></td><td></td><td>17</td><td>0.45</td><td>2.27</td><td></td></tr></tbody></table>

seconds apart) that the C<sub>3</sub> and C<sub>4</sub> series were poorly or not separated and that the identification was carried out by the mass spectrometer. He et al. with an Eclipse XDB C18 (5 µm, 150 × 4.6 mm) [36] and Machado et al. with a Novapak C18 (4 µm, 150 × 3.9 mm) [35] both reported that AO-DNPH and AC-DNPH coeluted (2 of the C<sub>3</sub>).

Sekine et al., while using a C<sub>18</sub> column with a similar dimension (Inertsil ODS-80A 5 µm, 150 × 4.6 mm), achieved a good resolution for the first six compounds analysed in our study (from FA-DNPH to CA-DNPH) [37]. They used 50:50 ACN/H<sub>2</sub>O in a 21-minute run. They focused on short-chain carbonyl compounds, using high water proportion in the eluant, leading to long retention times. In comparison, our method allowed the elution of CA-DNPH in about 8 min with a column of dimension of 3 µm, 250 × 3 mm and ACN/H<sub>2</sub>O at 75:25.

Another difference between the C<sub>18</sub> columns worth noting is the type of grafting or end-capping. Uchiyama et al. analysed the first 5 compounds of the same list, with complete separation of the C<sub>3</sub> critical trio [38]. As before, their method took more than 20 min to elute C<sub>3</sub> and PA-DNPH. They, however, used a C<sub>18</sub> column with amide grafting (2.7 µm, 150 × 4.6 mm). The use of a polar embedded column or the presence of polar end-capping groups might help discriminate the compounds thanks to displayed enhanced hydrogen bonding compared to traditionally end-capped phases [30].

The conditions used by Jiang et al. were the most like this work: although the column dimension was different (5 µm, 4.6 × 250 mm), it was also end-capped with a polar group and the same 13 compounds were analysed [19]. Unfortunately, no chromatogram, run duration or

<footnote><sup>a</sup> ODS: octadecylsilane</footnote>

<footnote><sup>b</sup> ACN: Acetonitrile, MeOH: Methanol</footnote>

<footnote><sup>c</sup> formaldehyde (FA), acetaldehyde (AA), acetone (AO), acrolein (AC), propionaldehyde (PA), crotonaldehyde (CA), methacrolein (MC), butanal (BA), butanone (BO), benzaldehyde (BZ) isovaleraldehyde (IA), valeraldehyde (VA), tolualdehyde (TA), hexanaldehyde (HA), methyl vinyl ketone (MVK)</footnote>

<footnote><sup>d</sup> with a guard cartridge.</footnote>

<footnote><sup>e</sup> compounds not analysed in this study.</footnote>

<footnote><sup>f</sup> other aldehyde or ketones, or oxoacids.</footnote>

<footnote><sup>g</sup> estimated from the available data.</footnote>

<footnote><sup>h</sup> DAD: Diode Array Detector.</footnote>
resolution values were reported.

Amongst the 13 compounds, our method did not allow the separation of BA-DNPH and BO-DNPH (2 of the C₄ compounds). In other methods, when they were analysed, usually no information about resolution was available. Only two studies also reported a coelution between BA-DNPH and BO-DNPH [23,39].

The column has a major influence on resolution, but also the pressure applied to the system. Due to the dimension of the column used in our work, a low flow rate set at 0.4 mL min⁻¹ had to be used to maintain reasonable pressure (~170 bar). Besides, in the field, minimising solvent consumption can be interesting to reduce waste to manage. With an elution time of 16 to 21 min for HA-DNPH depending on the HPLC used, only 6.8 to 8.4 mL of eluant is used. It makes our method the lowest eluant-consuming method, followed by Uchiyama et al. with 12 mL, ending the 0.6 mL min⁻¹ elution with PA-DNPH (5) [38]. All other methods used flow rates equal to or above 1.0 mL min⁻¹ for elution times in the same range or greater than in this work, thus consuming more eluant with volume in the range of 14–58 mL.

Lastly, LOD for formaldehyde-DNPH was listed in Table 3 in units of mg L⁻¹. To compare the sensitivity between the methods, the LOD was calculated in units of µg using the volume injected. The lowest LOD was 0.7 µg [19], followed by 0.12 µg (This work, HPLC B), then 0.17 µg [21, 22], and 0.60 µg, 1.09 µg, 2.27 µg (This work, HPLC C-UV, A and C-LED respectively), finishing with 49 µg [31].

To our knowledge and the extent of the information reported, no other isocratic method simultaneously achieved the separation of 11 of the 13 hydrazones with UV detection in less than 20 min and with a limit of detection below 1 µg for FA-DNPH. However, different HPLC systems were used, and other factors can reasonably influence the sensitivity and separation efficiency. For instance, the instability of the flow rate can increase the noise, and the ageing of the lamp can reduce the signal response, hence decreasing the sensitivity. Higher volume of detection cell, tubing, and the presence of dead volume will favour diffusion of the analytes and cause peak enlargement, reducing resolution and sensitivity.

### 3.3.2. Transportable HPLC

The above-mentioned systems were used for hydrazones of carbonyl compounds analysis, but all were laboratory benchtop HPLC systems. One of the main advantages of two HPLCs developed and tested in this publication is their transportability. To assess the non-chromatographic characteristics of the HPLC used in this study, BETTER criteria described by Rahimi et al. (2020) were used and plotted for each HPLC (see Fig. 6) [42]. Rahimi et al. defined a tool to facilitate objective comparison, to account for non-chromatographic characteristics (e.g., cost, weight, performance) and requirements for field-applicability systems (portability, robustness, operation time). BETTER grade levels range from 1 to 5 and represent the current range of developments, as well as community goals (see Table SI-7). Current portable HPLC systems reported in the literature or commercialised meet some of the grade 2 and 3 criteria [42,43]. Grades 4 and 5 represent challenging criteria to be met by the next generations, such as hand-portability, low cost, weeks of power autonomy and ability to withstand high pressure (>600 bar and >1300 bar). All these criteria are not compatible with each other yet. For instance, batteries able to supply enough power for several weeks would weigh too much for the system to be fully hand-portable. Also, for an HPLC to be able to work at high pressure, expensive elements such as high-pressure pumps or valves are required. Therefore, low cost and high performance (defined by BETTER as the ability to function at high pressure) are not compatible.

We assessed the non-chromatographic characteristics of the transportable HPLC C-UV and C-LED, and the benchtop HPLC A and B according to the BETTER criteria (Fig. 6, Table SI-8).

According to the chart, HPLC A/B and HPLC C differ in robustness and portability. HPLC C-UV and C-LED are resistant to transport and although it was not evaluated experimentally, we estimated that HPLC

<figure><img src="image_9.png" /><figcaption>Fig. 6. Radar chart assessing (a) HPLC A and B, and (b) HPLC C-UV and C-LED systems according to the BETTER criteria described by Rahimi et al. (2020).</figcaption></figure>

C-UV could not operate while in motion and HPLC C-LED could, thanks to its very simple detector configuration. These correspond to grade 2 and grade 3 for the robustness criterium. Careful transport is required for HPLC A/B. As for portability, HPLC C is defined as transportable using a standard vehicle (Grade 2). The already commercialised HPLC C-UV was taken on a plane a few times and dropped by airline company employees by inadvertence. The system functioned normally afterwards, and no damage was detected.

Other portable or transportable HPLCs and their characteristics are listed in Table 4. Our work focused on bringing a conventional HPLC to the field and it was achieved with components embedded in two highly robust suitcases: one analytical suitcase (23 kg) and one electronics/computer suitcase (19 kg). HPLC systems found in the literature were brought to a higher degree of miniaturisation. The weight of the lightest HPLC system reported was as low as 2 kg [53]. Ishida et al. combined a self-packed 800 µm i.d. column, an electroosmotic pump working at low pressure (40 bar max) and an LED-UV or conductivity detection. The lightest commercialised system weighs 8 kg [51] and utilizes a capillary column at a pressure up to 689 bar, syringes for pumping the eluant and UV-LED for detection.

Most of the miniaturised systems included a capillary column [28,29, 47–51]. Capillary liquid chromatography columns have the advantage of reducing solvent consumption due to their suitability to use low flow rates (in the nL/min and µL/min range). The analytical advantages also include a low amount of sample and enhanced sensitivity. The design of the instrument has to be thought carefully to be compatible with the use of such columns. The pump must be able to accurately deliver a flow in this range, and dead volumes that broaden peaks must be prevented. To do so, some authors even designed their system with “on-column” detection to reduce the peak widening effect [29,47]. The internal diameter of the other columns used was between 0.8 and 2.1 mm, and
<table><caption>**Table 4**
List of portable and transportable HPLC systems found in the literature.</caption><thead><tr><th>HPLC system</th><th>Weight</th><th>Column type (i. d.)</th><th>Pressure (max in bar)</th><th>Pump type Gradient: (g) Isocratic: (i)</th><th>Detection</th><th>Application</th><th>Reference</th></tr></thead><tbody><tr><td>MiliChrom-4<sup>a</sup></td><td>14 kg</td><td>Microbore (2 mm)</td><td>70</td><td>dual syringe (g)</td><td>two-beam multiwavelength spectrophotometer (190–360 nm)</td><td>pesticides, PAH, phenols, phtalate esters</td><td>[44]</td></tr><tr><td>MiLiChrom-6<sup>a</sup></td><td>17 kg</td><td>Microbore (2 mm)</td><td>70</td><td>dual syringe (g)</td><td>two-beam multiwavelength spectrophotometer (190–360 nm)</td><td>drugs, plants</td><td>[45,46]</td></tr><tr><td>Capillary gradient LC</td><td>1.3 kg (without electronics)</td><td>Capillary (100 µm)</td><td>116</td><td>4 syringes (g)</td><td>on-column LED-UV<sup>b</sup> (255, 355, 470, 600 nm)</td><td>dyes, preservatives</td><td>[29]</td></tr><tr><td>Nanoflow pumping system</td><td>4 kg (pump only)</td><td>Capillary (150 µm)</td><td>550</td><td>dual syringe (g)</td><td>on-column LED-UV<sup>b</sup> (260 nm)</td><td>phenols</td><td>[47]</td></tr><tr><td>EOP<sup>c</sup> HPLC cartridge</td><td>3 kg (without detector)</td><td>Capillary (75 µm)</td><td>140</td><td>electroosmotic pump (g)</td><td>VWD<sup>d</sup> or MS</td><td>peptide, proteins</td><td>[48]</td></tr><tr><td>Easy nanoLC<sup>e</sup> (+ Viking 573 MS*)</td><td>37 kg</td><td>Capillary (300 µm)</td><td>1200</td><td>dual syringe (g)</td><td>MS</td><td>opioids</td><td>[49].</td></tr><tr><td>Portable gradient capillary LC</td><td>2.7 kg</td><td>Capillary<sup>e</sup> (300 µm)</td><td>34</td><td>dual syringe (g)</td><td>LED-UV<sup>b</sup> (255 nm)</td><td>drugs</td><td>[50]</td></tr><tr><td>Axcend Focus<sup>b</sup> LC</td><td>8 kg</td><td>Capillary (300 µm)</td><td>689</td><td>dual syringe (g)</td><td>MS LED-UV<sup>b</sup> (235, 255, 275 nm)</td><td>trimethylxanthines, scopolamine, licit and illicit drugs</td><td>[51]</td></tr><tr><td>Capillary gradient LC</td><td>7.2 kg</td><td>Capillary (530 µm)</td><td>150</td><td>dual syringe (g)</td><td>LED-UV<sup>b</sup> (255 nm)</td><td>drugs</td><td>[28]</td></tr><tr><td>PAH portable LC</td><td>4.2 kg</td><td>Microbore (2.1 mm)</td><td>300</td><td>pre-compressed gas and a micro-valve (i)</td><td>spectrophotometer with pulsed xenon light source (180–890 nm)</td><td>PAHs</td><td>[52]</td></tr><tr><td>LC based on chip module</td><td>2 kg</td><td>Microbore<sup>e</sup> (0.8 mm)</td><td>40</td><td>electroosmotic pump (i)</td><td>LED-UV<sup>b</sup> (470 nm, 255 nm)</td><td>steroids, carotenoids, benzoate</td><td>[53]</td></tr><tr><td>HPLC C-UV<sup>a</sup></td><td>23 + 19 kg</td><td>Conventional (3 mm)</td><td>400</td><td>dual-piston pump (i)</td><td>Conductivity VWD<sup>d</sup> (190–500 nm)</td><td>Inorganic anions and cations carbonyl compounds</td><td>This work</td></tr><tr><td>HPLC C-LED</td><td>23 + 19 kg</td><td>Conventional (3 mm)</td><td>400</td><td>dual-piston pump (i)</td><td>LED-UV<sup>b</sup> (365 nm)</td><td>carbonyl compounds</td><td>This work</td></tr></tbody></table>

ours at 3 mm, making it the only conventional column in the transportable HPLC systems listed.

Rahimi et al. attributed the performance criteria to the ability of the system to withstand high pressures. To attain high pressures, the limiting factor is usually the type of pump used. Benchtop HPLC, as well as HPLC C in this work, are equipped with a piston pump, adapted to flow rates in the range of mL min⁻¹. Three other types of pumping systems were implemented in the literature: syringe pumps, electroosmotic pumps, and a pre-compressed gas pump. The syringe pumps use stepper-motor-driven syringes loaded with the eluant, delivering it gradually until it empties. One syringe allows isocratic elution and two can be used jointly to perform gradient analysis. The total run duration is limited by the volume of eluant the syringes can contain. To overcome this issue, Li et al. integrated two pairs of syringes connected to a 4-port switching valve, allowing two syringes to dispense mobile phase while the other two refill [29]. Lynch et al. and Ishida et al. used electroosmotic pumps capable of attaining pressures of 140 bar and 40 bar on their respective portable LC systems [48,53]. Electroosmotic pumps operate under a high electric field as a high voltage must be applied at the terminals of the capillary. The layer of ions at the charged surface migrates towards the oppositely charged electrode, thus creating a stable flow. A series of stacked electroosmotic pumps and valves allowed the creation of a gradient. However, electroosmotic pumps require high voltages, necessitating bulky external power supplies. On the other hand, Chatzimichail et al. described a pump operating without power. They designed a 6.7 kg HPLC system using a pre-pressurised gas container as mechanical power for pumping the mobile phase and reached a working pressure of up to 150 bars for flow rates in the nL min⁻¹ range. Using a

finite volume of gas may limit the number of runs possible when delivering in the mL min⁻¹ range (8–10 runs) but is not a limitation in the nL min⁻¹ range [52].

Performance cannot be solely described by the ability of the pump to generate high pressure. Another major component is the detection system. As the most universally used type of detector for HPLC, UV detection is naturally present in most miniaturised systems. To considerably reduce the weight and cost of this type of detector, a LED as a light source can be combined with a photodiode sensor [28,29,47,50,51, 53]. The number of applications is limited by the wavelength of the LED chosen. Other UV-Vis detectors offer expanded possibilities according to their detectable range of wavelengths [44–46,48,52]. A single (or a few in some cases) chosen wavelengths can be detected at once. It does not allow full scanning of the peaks and is therefore dedicated to targeted analysis.

Mass spectrometers offer a major advantage for the identification of compounds. However, their level of miniaturisation did not reach what was achieved with UV detection yet, as creating a low-cost and low-energy-consuming vacuum pump is a challenge [54]. Lynch et al. used a commercialised ESI-MS (Electrospray ionisation mass spectrometer) that was not miniaturised. Abonamah et al. used a Viking 573 transportable gas chromatograph-quadrupole mass spectrometer and obtained laboratory-quality data, using the NIST library for compound identification. Lam et al. integrated into their system a transportable quadrupole-based 4500 MiD mass spectrometer, equipped with a chip ESI source [48–50].

Although our system is heavy compared to the miniaturised systems weighing a few kilograms, it has been well thought out for on-field

<footnote>a Commercialised.</footnote>

<footnote>b Single Wavelength Detector using a Light Emitting Diode as a light source (LED-UV); Electro-Osmotic Pressure (EOP).</footnote>

<footnote>d Variable Wavelength Detector (VWD).</footnote>

<footnote>e in-house packed.</footnote>
analysis. The suitcase format (23 + 19 kg) makes it easy to transport, even in extreme and less-than-ideal shock-absorbing conditions such as those encountered when flying. In addition, the start-up can be done in an hour upon arrival. Furthermore, integrating conventional but robust components has the advantage of easing method transfer, especially when standardised methods are used.

## 4. Conclusion

In this study, an isocratic HPLC method for the analysis of 13 DNPH-derived carbonyl compounds applicable to a transportable HPLC was characterised, and the use of a developed transportable HPLC was validated through comparison with benchtop HPLCs. The isocratic method was based on the standard ISO 16,000-3 consisting of air sampling air through a DNPH tube for derivation of the carbonyl compounds into hydrazone, followed by elution with acetonitrile for a reverse-phase separation in an HPLC-UV. The transportable HPLC system was integrated into a reinforced suitcase and comprised an isocratic pump, a piston pump for sample delivery, and either a UV or LED detector. In comparison to other developed transportable or portable HPLC systems, it was simplified but not miniaturized further to ensure optimal performance.

13 hydrazones of aldehydes were analysed in isocratic mode in acetonitrile and water through a C₁₈ column. The run duration varied slightly between the systems but was less than 20 min long. Separation of the three C₃ and two of the four C₄ compounds was achieved. The separation of the second critical pair with 4 carbon atoms was conditional on a gradient elution and could not be achieved in isocratic conditions. After a literature study, we could safely conclude that our method was the first isocratic method to simultaneously achieve the separation of 11 of the 13 hydrazones with UV detection in less than 20 min and with a limit of detection below 1 µg for FA-DNPH. The LOD based on the height method for the hydrazones of the carbonyl compounds ranged from 0.12 to 0.38 mg L⁻¹ with the UV detector and 0.45 to 1.04 mg L⁻¹ with the LED detector, while LOD between 0.22 to 0.60 and 0.02 to 0.05 mg L⁻¹ were obtained the HPLC and UHPLC used as references. In terms of precision, RSD was calculated from the area obtained from 10 consecutive injections of the analytes, resulting in values of RSD<11.5% (UV) and RSD<14.1% (LED) when injecting concentration just above the detection limits. Performances were close to those of the laboratory HPLCs, either in sensitivity, resolution, or repeatability with overall better results with the UV than the LED detector.

Since the LED detector is not optically complex, it can be considered more robust than the UV detector. However, considering the higher LOD and poorer resolution values obtained, improvements can be made to achieve similar performances to the other HPLCs. The correlation coefficients of the linearities were below 0.999 with the LED detector and, therefore did not meet the standard procedure requirements. While our system may be comparatively heavy in contrast to miniaturized counterparts weighing a few kilograms reported in the literature, the suitcase format enhances portability, robustness in challenging conditions of transportation and fast set-up. The use of this HPLC for this application still requires a trained operator to manually elute the DNPH tubes. Future work will include a modification of the sample delivery system to perform automatic elution.

### CRediT authorship contribution statement

**Audrey Grandjean:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Conceptualization.
**Anaïs Becker:** Writing – review & editing, Methodology, Conceptualization.
**Mathilde Mascles:** Methodology, Formal analysis.
**Franck Amiet:** Resources, Funding acquisition.
**Jean-Philippe Amiet:** Resources, Funding acquisition.
**Damien Bazin:** Writing – review & editing, Validation, Supervision, Methodology,

Conceptualization. **Stéphane Le Calvé:** Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

This study was supported by the CIFRE programme between SME Chromatotec (Val-de-Virvée, France) and the French National Association of Research and Technology (ANRT, grant number 2020/0977).

We also thank Mr Mazet from Knauer for his help with the analytical columns.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jcoa.2024.100201.

### Data availability

Data will be made available on request.

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