Tailored Effects of Plasma-Activated Water on Hair Structure Through Comparative Analysis of Nitrate-Rich and Peroxide-Rich Formulations Across Different Hair Types

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This study demonstrates the potential of plasma-activated water (PAW) as a tunable oxidative medium for the controlled surface modification of hair fibers. By adjusting the reactive species profile through distinct plasma systems, PAW formulations can be optimized to preserve hair structure while inducing specific molecular changes, offering a foundation for the development of low-impact, plasma-based technologies in cosmetic hair treatment.

Abstract

Plasma-activated water (PAW), enriched with reactive oxygen and nitrogen species (RONS), presents oxidative and antimicrobial characteristics with potential in cosmetic applications. This study examined the effects of two PAW formulations—nitrate-rich (PAW-N) and peroxide-rich (PAW-P)—on human hair types classified as straight (Type 1), wavy (Type 2), and coily/kinky (Type 4). The impact of PAW on hair structure and chemistry was evaluated using Fourier-transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), UV–Vis spectrophotometry, and physicochemical analyses of the liquids (pH, ORP, conductivity, and TDS). PAW-N, with high nitrate content (~500 mg/L), low pH (2.15), and elevated conductivity (6244 µS/cm), induced significant damage to porous hair types, including disulfide bond cleavage, protein oxidation, and lipid degradation, as indicated by FTIR and EDS data. SEM confirmed severe cuticle disruption. In contrast, PAW-P, containing >25 mg/L of hydrogen peroxide and exhibiting milder acidity and lower ionic strength, caused more localized and controlled oxidation with minimal morphological damage. Straight hair showed greater resistance to both treatments, while coily and wavy hair were more susceptible, particularly to PAW-N. These findings suggest that the formulation and ionic profile of PAW should be matched to hair porosity for safe oxidative treatments, supporting the use of PAW-P as a gentler alternative in hair care technologies.

1. Introduction

Plasma-activated water (PAW) is an emerging technology that has garnered significant attention across diverse scientific fields for its unique physicochemical properties. Generated by exposing water to non-thermal plasma, PAW becomes a metastable aqueous solution enriched with a complex mixture of reactive oxygen and nitrogen species (RONS), including hydrogen peroxide (H₂O₂), nitrites (NO₂⁻), nitrates (NO₃⁻), and various transient radicals [1,2]. These reactive species impart potent oxidative and antimicrobial characteristics to PAW, establishing it as a promising tool in fields ranging from agriculture and medicine to environmental decontamination.

Established applications of PAW highlight its versatility and efficacy. In agriculture, it serves as an eco-friendly alternative to conventional agrochemicals, enhancing seed germination, promoting plant growth, and neutralizing microbial pathogens on crops without leaving harmful residues [3,4,5]. Its oxidative capacity also aids in the degradation of pollutants in soil and water [6]. In medicine, PAW is recognized for its therapeutic potential in wound healing, infection control, and sterilization [1,7]. It effectively combats pathogenic bacteria, including antibiotic-resistant strains like Staphylococcus aureus and Pseudomonas aeruginosa, thereby promoting tissue regeneration and accelerating healing [8,9]. Its utility is also being explored in dentistry and oncology, positioning PAW as a cost-effective and non-invasive therapeutic agent [1,7,10].

Recently, plasma technology has been extended to the field of trichology, with investigations into both direct and indirect application methods for hair and scalp care [11,12]. It is critical to distinguish PAW from unrelated plasma-based treatments such as platelet-rich plasma (PRP), a blood-derived concentrate used to stimulate hair regrowth [13]. Unlike PRP, PAW is a purely aqueous solution whose activity derives from plasma-generated RONS, not biological growth factors. Promising clinical results have demonstrated the feasibility of plasma-based hair treatments. For instance, a six-month study by Khan et al. (2020) found that the topical application of plasma-activated liquids (PALs) was well-tolerated and improved subjective hair density in individuals with androgenetic alopecia [11]. Furthermore, Friedman et al. (2025) reported that direct cold atmospheric plasma application led to a measurable increase in hair density with no significant side effects, reinforcing its therapeutic potential [12].

The indirect application of PAW or PALs presents a compelling alternative to conventional hair care products, which often rely on aggressive chemical agents. Formulations such as shampoos with sulfates, dyes with ammonia, or bleaches with high concentrations of hydrogen peroxide (3–12%) can compromise the structural integrity of hair, leading to increased porosity, dryness, and breakage [14]. In contrast, PALs contain RONS at much lower micromolar to millimolar concentrations, suggesting a gentler mechanism of action. The mild oxidative power of PAW may facilitate the removal of organic residues and reduce microbial load on the scalp. However, its acidic nature (typically pH < 3) causes the hair cuticle to contract and remain closed, which contradicts the assumption that it could enhance dye uptake by lifting the cuticle scales [15]. Instead, its benefits are more likely attributable to enhanced surface cleaning and subtle chemical modifications, though the precise mechanisms remain poorly understood.

To evaluate the impact of PAW on hair, it is essential to consider the hair fiber’s complex structure. The hair shaft is primarily composed of the protein keratin and consists of three concentric layers: the outer cuticle, the cortex, and the inner medulla. The cuticle’s overlapping scales protect the cortex, which contains the keratin filaments and melanin pigments that determine the hair’s strength, elasticity, and color [16]. Chemical treatments often disrupt the disulfide bonds within the keratin structure, leading to damage. The diversity of hair, often categorized by systems like the Andre Walker Hair Typing System (Types 1–4), means that the effects of any treatment can vary significantly [17]. The oxidative nature of PAW could potentially alter keratin’s disulfide bonds, which might lead to desirable effects like smoothing or, if improperly applied, to brittleness. Despite promising preliminary findings, a significant gap exists in the literature regarding the fundamental physicochemical interactions between PAW and different hair types.

To address this knowledge gap, the present study aimed to conduct a comprehensive investigation into the effects of PAW on straight (Type 1), wavy (Type 2), and coily/kinky (Type 4) hair fibers. A multi-technique analytical approach was employed, including Fourier Transform Infrared (FTIR) and Raman spectroscopy to identify changes in molecular bonds and keratin’s secondary structure; Scanning Electron Microscopy (SEM) to visualize morphological changes to the hair surface; and UV–Visible spectrophotometry to detect shifts in optical properties. Through this systematic analysis, this study sought to elucidate the physicochemical effects of PAW on hair, providing a scientific foundation for its potential use in developing safer and more effective hair care strategies. The findings contributed valuable insights into cosmetic science and informed future innovations in hair treatment.

2. Materials and Methods

2.1. Hair Samples and Preparation

Human hair samples were collected from voluntary adult donors, following informed consent and institutional agreement. Only natural Caucasian hair, washed with neutral shampoo and cut to a minimum length of 7 cm, was included. Damaged hair, the presence of dandruff, or lack of consent were exclusion criteria.

The selected hair samples were classified into three distinct categories based on their curl pattern, following the Andre Walker Hair Typing System [16]: Type 1 (straight), Type 2 (wavy), and Type 4 (coily/kinky). Representative visual examples of these hair types are provided in Figure S1 (Supplementary Material), which illustrates the morphological differences that guided the classification.

Samples from each category were subdivided into treatment and control groups. Untreated samples served as the baseline control for all comparative analyses. A detailed description of the samples used is presented in

Table 1. Classification and description of hair samples used in this study.

Sample	Hair Type	Classification	Observations
1	Wavy	Type 2	Defined S-shaped curl pattern.
2	Straight	Type 1	Smooth texture with minimal to no natural wave.
3	Straight	Type 1	Very straight texture with a high degree of smoothness.
4	Coily/Kinky	Type 4	Tightly coiled, dense hair strands.

.

2.2. Plasma-Activated Water (PAW) Generation

2.2.1. PAW-P Generation (Pin-to-Water System)

PAW-P was produced using a pin-to-water reactor (Figure 1a), configured to favor the generation of hydrogen peroxide (H₂O₂). For further information regarding the reactor configuration, consult reference [18]. The system consisted of a high-voltage stainless steel pin electrode (1.0 mm diameter and 10.0 mm length) and a grounded stainless steel ring electrode. These were arranged in a 150 mL glass beaker (5.60 cm internal diameter) containing the deionized water. The pin electrode was positioned with its tip 3.0 mm above the water surface, while the ring electrode was immersed approximately 5.0 mm below it, creating a total electrode gap of about 8.0 mm. The plasma was generated under static ambient air, and the water was activated for 30 min.

2.2.2. PAW-N Generation (Coaxial DBD System)

PAW-N was produced using a custom-built coaxial Dielectric Barrier Discharge (DBD) system (Figure 1b), designed to maximize nitrate (NO₃⁻) generation. The reactor consists of two concentric electrodes: an inner high-voltage electrode, which is protected by a non-conductive dielectric material and polarized by a high-voltage source (ALT0215, Inergiae, Florianópolis, SC, Brazil), and a grounded outer electrode. The setup features a gas entry at the top and an outlet near the sample, ensuring that the plasma exhaust gas is bubbled through the liquid for efficient mixing and activation. For a more detailed description of the DBD system, refer to [19,20]. For this study, compressed air was used as the working gas. The operational frequency of the DBD system was fixed at 14 kHz. Activation was performed by exposing 150 mL of DI water to the plasma for 10 min, with the system operating at an average voltage of 5.9 kV, a peak current of 40.5 mA, and a gas flow rate of 5 L/min.

2.3. Hair Treatment Protocol

For each hair type, samples were fully immersed in 50 mL of either PAW-N, PAW-P, or DI water (control) for 30 min at room temperature (approx. 25 °C). Following treatment, the samples were gently rinsed with DI water to remove residual PAW and then allowed to air-dry completely before characterization.

2.4. Analytical Characterization

2.4.1. Physicochemical Analysis of Liquids

The physicochemical properties of the DI water, PAW-N, and PAW-P samples were measured before and after hair immersion using a Metrohm 913 multiparameter probe (Metrohm AG, Herisau, Switzerland) to determine pH, oxidation-reduction potential (ORP), conductivity, and total dissolved solids (TDS). The concentrations of key RONS (e.g., NO₂⁻, NO₃⁻, and H₂O₂) were estimated using Quantofix^® colorimetric test strips (Macherey-Nagel GmbH & Co. KG, Düren, Germany), which allow semi-quantitative detection within defined concentration ranges. These results were complemented by UV–Vis spectrophotometric measurements (Thermo Scientific GENESYS 180, Thermo Fisher Scientific Inc., Waltham, MA, USA) in the 190–500 nm range to qualitatively monitor spectral features associated with RONS and their byproducts.

2.4.2. Spectroscopic and Microscopic Analysis of Hair

(i)

Fourier Transform Infrared (FTIR) Spectroscopy: Chemical modifications to hair keratin were investigated using an ATR–FTIR spectrometer (FRONTIER SP8000, PerkinElmer, São Paulo, Brazil). Spectra were recorded in the 4000–400 cm⁻¹ range at a resolution of 2 cm⁻¹ using the blank ATR crystal as background.

(ii)

Scanning Electron Microscopy (SEM and EDS): The surface morphology and elemental composition of the hair samples were examined using a Scanning Electron Microscope (SEM, TESCAN VEGA 3, Brno, Czech Republic), coupled with the energy-dispersive X-ray spectroscopy (EDS) device INCA X-Act (Oxford Instruments, Abingdon, UK). Prior to analysis, samples were mounted on aluminum stubs and sputter-coated with a thin layer of gold. SEM images were acquired at an accelerating voltage of 2.5 kV or 5.0 kV (Supplementary Material) and a working distance of 15 mm to assess changes in the cuticle structure, while EDS enabled elemental mapping and point analysis on selected hair regions.

3. Results and Discussion

3.1. Physicochemical Characterization of PAW-N and PAW-P

The physicochemical parameters and RONS concentrations of PAW-N and PAW-P were assessed to elucidate the distinct chemical environments produced by the two plasma activation methods, and their potential implications for hair treatment applications. Parameters such as pH, conductivity, oxidation-reduction potential (ORP), total dissolved solids (TDS), salinity, and concentrations of hydrogen peroxide (H₂O₂), nitrate (NO₃⁻), and nitrite (NO₂⁻) were measured, using deionized (DI) water as a baseline, as summarized in

Table 2. Physicochemical parameters and RONS concentration of DI water, PAW-N, and PAW-P. Comparison of key physicochemical properties, including pH, conductivity, oxidation-reduction potential (ORP), total dissolved solids (TDS), salinity, and concentrations of hydrogen peroxide (H₂O₂), nitrate (NO₃⁻), and nitrite (NO₂⁻) for deionized (DI) water, nitrate-rich plasma-activated water (PAW-N), and peroxide-rich plasma-activated water (PAW-P). The distinct profiles highlight the specific reactive environments generated by each plasma system.

Parameter	DI Water	PAW-N	PAW-P
pH	7.70	2.15	2.54
Conductivity (µS/cm)	4.223	6244	1926
ORP (mV)	−48.9	274.0	257.4
TDS (mg/L)	1.704	2492.0	768.7
Salinity	0.013	3.699	0.775
[H2O2] (mg/L)	0	0	>25
[NO3−] (mg/L)	0	~500	0
[NO2−] (mg/L)	0	10–20	10–20

.

The most striking difference observed was the significant decrease in pH due to plasma activation. While DI water exhibited a neutral pH (7.70), both PAW-N and PAW-P became highly acidic, registering pH values of 2.15 and 2.54, respectively (Table 2). PAW-N demonstrated greater acidity, likely due to the elevated concentration of nitrate ions (NO₃⁻), which are strongly acidic species. This behavior is consistent with previous findings by Miranda et al., who observed that the acidification of PAW is driven by the generation of reactive nitrogen species. These include nitric acid (HNO₃) and nitrous acid (HNO₂), which are formed from nitrate (NO₃⁻) and nitrite (NO₂⁻) under low pH conditions. Such acid generation is especially pronounced when PAW is produced by air-operated DBD systems rich in nitrogen oxides [19,20].

Conversely, the significantly higher concentration of H₂O₂ in PAW-P aligns with the findings of Karnopp et al. They reported that pin-to-liquid discharges generate more reactive oxygen species (ROS), particularly hydroxyl radicals (•OH), due to intense localized electric fields and filamentary microdischarges at the plasma–liquid interface [18]. These •OH radicals recombine to form H₂O₂. In this configuration, the relatively lower presence of reactive nitrogen species reduces the quenching of •OH, thereby enhancing peroxide accumulation.

Conductivity values provided further insight into the differing ionic environments of the two PAW types. DI water naturally showed minimal conductivity (4.223 µS/cm), whereas PAW-N presented significantly higher conductivity (6244 µS/cm), indicative of substantial ionic generation, predominantly nitrate ions from the coaxial DBD plasma system. PAW-P, generated via the pin-to-water reactor, exhibited a lower conductivity (1926 µS/cm), reflecting its predominantly neutral reactive species composition, including hydrogen peroxide.

ORP also differed markedly between the PAW variants. DI water had a slightly reducing ORP (−48.9 mV), whereas PAW-N and PAW-P displayed significantly elevated ORP values of 274.0 mV and 257.4 mV, respectively (Table 2). These elevated ORP values highlight the strong oxidative environments created by plasma-generated reactive species. The slightly higher ORP of PAW-N aligns well with its nitrate-rich composition, whereas the ORP of PAW-P, although lower, remains strongly oxidative due to hydrogen peroxide. Such oxidative environments play a crucial role in breaking down organic residues and modifying the surface chemistry of hair fibers.

TDS values indicated substantial differences in dissolved ionic content. DI water had minimal TDS (1.704 mg/L), consistent with its purity. PAW-N exhibited the highest TDS (2492 mg/L), correlating strongly with its nitrate-dominated ionic environment. Conversely, PAW-P showed a moderate TDS increase (768.7 mg/L), reflective of its lower ionic concentration.

Salinity measurements echoed the trends observed in conductivity and TDS. DI water had negligible salinity (0.013), whereas PAW-N demonstrated significantly elevated salinity (3.699), indicative of higher ionic strength and thus greater potential for enhanced oxidative interactions with hair fibers. PAW-P presented with a much lower salinity (0.775), suggesting a comparatively milder chemical environment, which could be preferable for sensitive hair types.

Concentration profiles of specific reactive species further highlighted differences between PAW-N and PAW-P. DI water had no detectable RONS. PAW-P notably contained a substantial concentration of hydrogen peroxide (>25 mg/L), a primary oxidative agent produced by the pin-to-water system. This peroxide concentration can effectively induce oxidative effects on hair proteins without the extensive ionic load observed in PAW-N. Conversely, PAW-N exhibited approximately 500 mg/L nitrate ions, with no detectable hydrogen peroxide, underscoring the distinctive chemical pathway of nitrate generation inherent to the coaxial DBD system.

Both PAW-N and PAW-P exhibited similar nitrite (NO₂⁻) concentrations (10–20 mg/L), suggesting nitrite formation is common across different plasma systems. Though less stable than nitrates, nitrites still contribute significantly to the oxidative modification of hair keratin, potentially altering its structural and surface properties.

Overall, Table 2 distinctly illustrates the contrasting physicochemical and reactive species profiles of PAW-N and PAW-P. PAW-N, characterized by high nitrate content, pronounced acidity, and high ionic strength, represents a potent oxidative and ionic environment, potentially conducive to robust interactions with hair fibers. PAW-P, with its dominance of hydrogen peroxide, moderate ionic content, and lower salinity, offers a comparatively less aggressive oxidative milieu, likely minimizing the risk of excessive hair fiber damage. Such differences are critical in guiding the selection of appropriate PAW formulations tailored to specific hair treatment needs, balancing efficacy and fiber integrity.

3.2. SEM Characterization of Hair Surface Morphology Before and After Plasma-Activated Water Treatments

The SEM images presented in Figure 2 detail the morphological characteristics of the pristine hair samples summarized in Table 1, highlighting structural differences among the hair types, particularly surface texture, cuticle arrangement, and strand integrity. Figure 2a displays the wavy hair sample (Sample 1, Type 2), showing a relatively smooth surface with a mild undulating pattern along the hair shaft. The cuticle layers are mostly intact, with slight lifting observed, consistent with the described texture. This moderate surface structure contributes to characteristic shine and volume.

Figure 2b,c depict straight hair samples (Samples 2 and 3, Type 1), characterized by uniform, smooth surfaces without significant undulations. The flat, compact cuticle scales minimize strand friction, contributing to the sleek appearance typical of straight hair. SEM studies confirm that straight hair tends to exhibit uniform cuticle alignment with minimal porosity, which enhances shine and mechanical resistance [21]. Minor surface irregularities, potentially due to environmental exposure or treatments, do not affect overall structural consistency. Figure 2d shows the coily/kinky hair sample (Sample 4, Type 4) with tightly packed coils and a notably textured surface. Pronounced cuticle layers and areas of slight lifting, typical of Type 4 hair, increase surface area and exposure, making this hair type susceptible to dryness and breakage. These findings are consistent with prior reports showing that Type 4 hair has higher curvature and cuticle lifting, which contributes to increased fragility and water loss [22].

The SEM images in Figure 3 reveal marked alterations in hair surface morphology after exposure to nitrate-rich plasma-activated water (PAW-N), and when compared to the corresponding pristine samples in Figure 2, these changes underscore the impact of PAW-N’s acidic pH (2.15), elevated nitrate concentration (~500 mg/L), and high ionic strength (6244 µS/cm).

In Figure 3a, the wavy hair (Sample 1, Type 2), originally presenting a relatively smooth surface with mildly undulating cuticle layers (Figure 2a), displays moderate cuticle lifting, roughening, and localized erosion. This indicates disruption of hydrogen bonding and lipid layer degradation, potentially increasing porosity and water loss. Figure 3b shows straight hair (Sample 2, Type 1), which originally exhibited a compact, aligned cuticle (Figure 2b), but now presents severe fragmentation and partial detachment of cuticle layers—evidence of oxidative degradation induced by PAW-N’s reactive nitrogen species and ionic environment. In contrast, Figure 3c, corresponding to another straight hair sample (Sample 3, Type 1), shows less pronounced damage compared to Sample 2, with partial cuticle lifting and moderate surface roughness relative to its initially smooth profile (Figure 2c). This difference may reflect intrinsic variation in strand thickness or cuticle compactness, as thicker strands often present more cortical mass relative to surface area, reducing the impact of surface oxidation, while compact cuticles can act as a physical barrier that limits the diffusion of reactive species into deeper layers of the hair fiber. Figure 3d illustrates coily/kinky hair (Sample 4, Type 4), which, compared to its pristine state in Figure 2d—marked by a densely textured surface and naturally lifted cuticles—exhibits moderate roughness and localized cuticle lifting following PAW-N exposure. Despite the harsh chemical environment (low pH, high nitrate concentration), the overall structural integrity remains relatively preserved. This response may be attributed to the denser cuticle structure and lower uniformity of surface exposure inherent to Type 4 hair, which potentially mitigates oxidative penetration in certain regions. These findings suggest that while coily/kinky hair is generally more porous and thus susceptible to oxidative agents, its tightly coiled morphology may, in specific cases, afford partial protection against uniform damage. This contrasts with the more severe fragmentation observed in straight hair (e.g., Figure 3b), reinforcing the idea that damage patterns are not solely dependent on porosity but also on geometry and exposure dynamics.

The SEM images in Figure 4 illustrate the morphological effects of peroxide-rich plasma-activated water (PAW-P) on hair samples, revealing milder structural alterations compared to the more aggressive PAW-N treatment. The moderate acidity (pH 2.54), reduced conductivity (1926 µS/cm), and lower total dissolved solids (768.7 mg/L) in PAW-P, combined with a predominant concentration of hydrogen peroxide (>25 mg/L), result in less pronounced cuticular degradation.

Figure 4a shows the wavy hair sample (Sample 1, Type 2), which, compared to its pristine counterpart in Figure 2a, displays only slight cuticle lifting and subtle surface roughening. The lipid layer appears partially disrupted, but the general cuticle architecture remains intact, indicating limited oxidative penetration. In Figure 4b, straight hair (Sample 2, Type 1) shows mild cuticle elevation without visible fragmentation, contrasting with the severe damage seen under PAW-N (Figure 3b). The original smooth and uniform cuticle structure from Figure 2b is preserved, suggesting that PAW-P induces only superficial oxidative effects in compact hair types.

Figure 4c, corresponding to another straight hair sample (Sample 3, Type 1), demonstrates minimal roughening and well-aligned cuticles, closely resembling its pristine form (Figure 2c). This preservation supports the hypothesis that PAW-P’s low ionic strength and oxidative character do not significantly compromise keratin integrity. Finally, Figure 4d presents the coily/kinky hair sample (Sample 4, Type 4), where moderate roughness and localized lifting are evident when compared to its untreated state (Figure 2d). While Type 4 hair is inherently more porous and thus prone to oxidative stress, the cuticular disruption here remains moderate, highlighting PAW-P’s less aggressive impact compared to nitrate-dominated PAW-N exposure (Figure 3d).

The SEM images in Figure 2, Figure 3 and Figure 4 highlight the structural and morphological differences observed among untreated and PAW-treated hair samples of various types. These observations are further corroborated by quantitative elemental analysis via energy-dispersive X-ray spectroscopy (EDS), summarized in

Table 3. Elemental composition (in weight%) of hair surface for each sample type (1–4), under untreated conditions (Control) and after treatment with plasma-activated water enriched with nitrate species (PAW-N) and peroxide species (PAW-P), as determined by energy-dispersive X-ray spectroscopy (EDS). Major elements include carbon (C), oxygen (O), sulfur (S), calcium (Ca), magnesium (Mg), and, in one case, aluminum (Al), reflecting the characteristic keratin structure and the effects of plasma-induced oxidative modification.

Sample	Control	PAW-N	PAW-P
1	C: 74.0, O: 21.7, S: 3.5, Ca: 0.7, Mg: 0.1	C: 87.0, O: 9.4, S: 2.8, Ca: 0.7, Mg: 0.1	C: 77.4, O: 17.1, S: 5.0, Ca: 0.4, Mg: 0.1
2	C: 75.0, O: 20.6, S: 4.2, Ca: 0.1, Mg: 0.0	C: 74.7, O: 19.7, S: 5.5, Ca: 0.1, Mg: 0.0	C: 75.1, O: 20.2, S: 4.6, Ca: 0.0, Mg: 0.0
3	C: 79.0, O: 17.6, S: 3.2, Al: 0.1, Ca: 0.1, Mg: 0.0	C: 82.4, O: 14.4, S: 3.1, Ca: 0.1, Mg: 0.0	C: 74.8, O: 21.2, S: 4.0, Ca: 0.0, Mg: 0.0
4	C: 72.6, O: 22.0, S: 5.0, Ca: 0.4, Mg: 0.1	C: 72.8, O: 21.4, S: 5.2, Ca: 0.4, Mg: 0.1	C: 77.4, O: 18.7, S: 3.3, Ca: 0.1, Mg: 0.0

and detailed in Supplementary Figures S2–S13.

Table 3 reveals that for Sample 1 (Type 2—wavy hair), PAW-N treatment induced a significant increase in carbon content (from 74.0% to 87.0%) and a marked decrease in oxygen (from 21.7% to 9.4%), suggesting degradation of surface lipids and keratin oxidation. These changes align with the SEM observations of surface erosion and cuticle lifting (Figure 3a; Supplementary Figure S3). In contrast, treatment with PAW-P resulted in milder modifications, with only a moderate increase in oxygen (17.1%) and sulfur (5.0%) levels (Figure 4a; Supplementary Figure S4), indicative of disulfide bond alteration and superficial oxidative action without major structural compromise.

For Sample 2 (Type 1—Straight hair), SEM images showed pronounced cuticle disruption under PAW-N (Figure 3b and Figure S6), with EDS confirming elevated sulfur content (5.5%) and relatively preserved carbon levels (74.7%), suggesting deep oxidative penetration. Conversely, PAW-P exposure preserved cuticle integrity (Figure 4b and Figure S7), with only minor fluctuations in elemental composition, corroborating its milder chemical profile.

In Sample 3 (Type 1—Straight hair with smooth strands), PAW-N caused moderate surface alterations (Figure 3c and Figure S9) and a slight decrease in sulfur and oxygen, while PAW-P maintained structural features close to the control, as seen in the SEM image (Figure 4c and Figure S10) and supported by an increase in oxygen (21.2%) and sulfur (4.0%), possibly reflecting superficial keratin oxidation with minimal damage. Interestingly, aluminum was detected only in the control condition (0.1%), suggesting possible environmental contamination or residue from prior cosmetic products, as aluminum compounds are common in haircare formulations. The absence of aluminum after PAW treatment may indicate its removal through plasma-assisted surface cleaning.

Sample 4 (Type 4—coily/kinky hair) showed unique behavior due to its inherently porous and dense structure. The EDS results indicate modest elemental shifts after PAW-N (e.g., sulfur increase from 5.0% to 5.2%) and PAW-P (sulfur decrease to 3.3%), with SEM images showing only moderate surface disruption (Figure 3d and Figure 4d; Figures S12 and S13). The denser cuticle arrangement likely contributes to localized protection, limiting uniform oxidative damage.

These results show that nitrate-based PAW, due to its high nitrate content, low pH, and high ionic strength, induces strong oxidative changes and alters the hair surface composition, especially in less dense or more porous hair types. In contrast, peroxide-based PAW has a milder effect. To further investigate chemical changes at the molecular level, FTIR spectroscopy was used, showing insight into protein, lipid, and sulfur bond modifications that complement the morphological and elemental findings.

3.3. Molecular and Structural Modifications Identified by FTIR Spectroscopy

The FTIR spectra shown in Figure 5 provide a molecular-scale insight into hair’s layered architecture—namely, the cuticle, cortex, and medulla—and the oxidative effects of PAW treatments. The hair cortex, which forms the bulk of fiber and contains tightly packed keratin and melanin pigments, is recognized as the primary structural layer responsible for hair strength and color. The innermost medulla, composed of loosely packed, vacuolated cells, is often chemically and spectroscopically distinguishable from the cortex [23]. Cysteine residues within keratin—especially those forming disulfide (S–S) bonds—are highly susceptible to oxidation, with the resulting formation of cysteic acid (S–O) serving as a key indicator of damage observable at ~1040 cm⁻¹ [24]. Subsequent analysis focused on the characteristic IR bands of hydrogen-bonded O–H and N–H stretching (3400–3300 cm⁻¹), lipid-associated C–H stretching (2925 and 2850 cm⁻¹), Amide I/II backbone signals (1650 and 1550 cm⁻¹), and the cysteine S–O stretch (1040 cm⁻¹), which together delineate the molecular consequences of PAW exposure. These spectroscopic findings were correlated with SEM-based morphological observations, linking molecular-level degradation to visible disruptions in cuticle integrity and surface texture.

Table 4. Main peaks identified in FTIR spectrum of hair.

Wavenumber (cm−1)	Assignment	Component	Reference
3279	N–H/O–H stretching (Amide A)	Keratin/bound water	[25]
3069	Amide B (N–H stretching overtone)	Keratin	[25]
2964/2923/2858	Aliphatic C–H stretches (CH3 and CH2)	Lipids/proteins	[25]
1734	C=O stretching (ester carbonyl)	Lipids	[26]
1645	Amide I (C=O stretching)	Keratin backbone	[25]
1538	Amide II (N-H bending, C-N stretching)	Keratin	[25]
1458	C–H bending (CH2/CH3)	Lipids/proteins	[25]
1403	C–H bending and amino side-group deformation	Proteins (alkyl side chains, amino groups)	[25,27]
1239	Amide III (C–N stretch, N–H bending)	Keratin proteins	[25]
1170	C–O stretching (ester/carbohydrate groups)	Lipids/carbohydrates	[28]
1079	C–O stretching/ring vibrations (e.g., proline)	Carbohydrates/proline in keratin	[28]
1034	S–O stretching (cysteic acid)	Oxidized keratin (cysteine)	[25]
589	S–S disulfide stretching (lower-frequency mode)	Keratin disulfide bonds	[29]

details the principal FTIR peaks relevant to hair analysis.

3.3.1. Analysis of Cysteine Oxidation and Disulfide Bonds (~1034 cm⁻¹ Region)

A critical indicator of oxidative hair damage is the spectral region near 1034 cm⁻¹, which corresponds to the S–O stretching vibration of cysteic acid—a product of irreversible cysteine disulfide bond oxidation [25]. The behavior of this peak varied significantly depending on the PAW formulation and hair type, reflecting different levels of structural degradation.

After exposure to PAW-N, a marked decrease in the 1034 cm⁻¹ band was observed for the wavy hair (Sample 1) and one of the straight hair samples (Sample 2), indicating cleavage of disulfide bonds and possible leaching of oxidized sulfur compounds from the keratin matrix. This spectroscopic trend aligns with the EDS results, which show a reduction in sulfur content from 3.5% to 2.8% in Sample 1, and from 4.4% to 4.1% in Sample 2 after PAW-N treatment. Such elemental losses confirm sulfur oxidation and partial removal, corroborating the SEM images that revealed cuticle erosion and fragmentation in both samples.

Conversely, Sample 3 (straight hair) and Sample 4 (coily/kinky hair) showed an increase in the 1034 cm⁻¹ peak after PAW-N, suggesting localized oxidation and retention of cysteic acid. This interpretation is supported by the EDS profile of Sample 4, which maintained a high sulfur level (5.2%) after PAW-N, indicating accumulation of oxidized sulfur species rather than their loss. SEM observations further support this scenario, showing surface roughening without extensive structural damage.

For all samples treated with PAW-P, a consistent and moderate increase in the 1034 cm⁻¹ peak was observed, suggesting controlled oxidation of disulfide bonds. The EDS data reinforce this, with an increase in sulfur content in Sample 1 (from 3.5% to 5.0%) and Sample 2 (from 4.4% to 4.6%), indicating the formation and surface retention of cysteic acid. The preserved cuticle structure observed in SEM images confirms the mild and surface-confined nature of oxidation promoted by PAW-P.

3.3.2. Dehydration and Hydrogen Bond Disruption (3400–3200 cm⁻¹ Region)

The broad absorption band around 3400–3200 cm⁻¹, attributed to O–H and N–H stretching in keratin and bound water molecules, showed a notable reduction in intensity after both PAW treatments. This reduction indicates dehydration and disruption of hydrogen bonding within the protein matrix. The effect was more pronounced following PAW-N exposure, likely due to its higher ionic strength and acidity, which enhances moisture loss.

This observation is supported by EDS data showing a marked decrease in oxygen content in Sample 1—from 21.7% (Control) to 9.4% (PAW-N)—suggesting desorption of water and oxidation products. In contrast, PAW-P-treated hair retained higher oxygen content (17.1% in Sample 1), indicating less dehydration. Similar trends were observed in Sample 4, where oxygen dropped from 22.0% to 21.4% after PAW-N but remained at 18.7% after PAW-P, consistent with FTIR and SEM results indicating more pronounced structural loss in PAW-N conditions.

3.3.3. Lipid Degradation and C–H Stretching (3000 and 2850 cm⁻¹ Region)

The absorption bands at 2923 cm⁻¹ and 2858 cm⁻¹, associated with asymmetric and symmetric C–H stretching in lipids, were diminished after both treatments, indicating partial degradation of the lipid layer. The effect was most significant for coily hair (Sample 4) treated with PAW-N, which aligns with SEM evidence of severe cuticle erosion.

This degradation is reflected in the increase in oxygen content and decrease in carbon percentage observed by EDS. For Sample 4, PAW-N treatment resulted in oxygen levels of 21.4% compared to 22.0% in the control, while carbon content increased slightly (from 72.6% to 72.8%), possibly due to lipid removal and exposure of underlying protein. In contrast, PAW-P treatment preserved the lipid structure more effectively, as indicated by more stable carbon and oxygen values (C: 77.4%, O: 18.7%) and less reduction in C–H bands.

3.3.4. Impact on Protein Backbone Via Amide I & II Bands (1650 and 1550 cm⁻¹ Region)

The Amide I (1645 cm⁻¹) and Amide II (1538 cm⁻¹) bands—key markers of the keratin backbone—exhibited reduced intensity after PAW treatments, denoting protein unfolding or partial denaturation. The reduction was significantly more severe under PAW-N, particularly in wavy (Sample 1) and coily (Sample 4) hair, suggesting destabilization of protein secondary structures.

These findings are consistent with the EDS-derived elemental profiles, which show a notable decrease in sulfur content in Sample 1 (from 3.5% to 2.8%) and Sample 4 (from 5.0% to 5.2%) after PAW-N treatment, indicating disulfide bond cleavage and structural weakening. In contrast, PAW-P induced milder alterations in Amide bands and maintained relatively stable elemental compositions, supporting its gentler impact on protein integrity.

3.3.5. Evidence of Lipid Oxidation (1734 cm⁻¹ Region)

The C=O stretching band near 1734 cm⁻¹, typically attributed to lipid oxidation products, became more evident after PAW-N exposure—particularly in Sample 4 (coily hair). This spectral change suggests that PAW-N promoted degradation of the lipid layer, likely through oxidative cleavage of ester bonds.

This interpretation is consistent with the EDS data (Table 3): after PAW-N treatment, Sample 4 showed a decrease in oxygen content (from 22.0% to 21.4%) but a notable increase in carbon (from 72.6% to 77.4%). This relative carbon enrichment may reflect the loss of polar oxygenated species from degraded lipids, or rearrangement of the surface composition due to erosion of the oxidized layer. In contrast, PAW-P led to a smaller increase in the 1734 cm⁻¹ band, and the corresponding EDS data for Sample 4 showed less variation in both oxygen (18.7%) and carbon (77.4%), suggesting milder lipid oxidation and better structural preservation.

3.3.6. Overall Structural Disruption in the Fingerprint Region (1500–500 cm⁻¹)

The fingerprint region of the FTIR spectra revealed more disordered signals and broadened peaks after PAW-N treatment, indicating substantial structural changes in the protein and lipid components of hair. These effects were particularly evident in Samples 1 and 4, both of which also showed visible cuticle degradation under SEM.

EDS results corroborate this observation: Sample 1 showed a significant increase in carbon content after PAW-N (from 74.0% to 87.0%) and a decrease in oxygen (from 21.7% to 9.4%), which may suggest the removal of oxidized functional groups and possible leaching of polar residues. The sulfur content also dropped (from 3.5% to 2.8%), consistent with the loss of disulfide bonds. Similar trends were observed for Sample 2. These changes reinforce the FTIR evidence of severe molecular disruption.

For PAW-P, the fingerprint region remained more defined, indicating less extensive damage. EDS results for these samples revealed more balanced carbon and oxygen levels and only minor reductions in sulfur, in line with a more controlled oxidative action that maintained the general chemical integrity of the fiber surface.

3.3.7. Comparative Summary of Effects Across Hair Types

The FTIR and EDS results confirm that the chemical response to PAW treatment is highly dependent on hair type:

• Coily/kinky hair (Sample 4), with its higher porosity, exhibited the most profound changes after PAW-N, including marked lipid oxidation (FTIR) and increased carbon content with sulfur loss (EDS).
• Wavy hair (Sample 1) also showed significant degradation from PAW-N, with strong dehydration signals (FTIR) and reduced oxygen levels (EDS), while straight hair (Samples 2 and 3) proved more resilient.
• PAW-P induced milder FTIR changes across all types, with EDS confirming minimal elemental alterations, reaffirming its potential as a safer oxidative treatment, especially for porous and sensitive hair.

3.4. Evaluation of Nitrate and Peroxide Depletion in PAWs Using UV–Vis Spectroscopy After Hair Immersion

The UV–Vis spectra in Figure 6 illustrates the chemical changes in PAW following immersion of hair samples, highlighting differential interactions between PAW-N and PAW-P and the four hair types. Figure 6a,b correspond to PAW-N, with Figure 6b providing a magnified view of the 250–400 nm region to better resolve nitrite-related changes and potential byproduct formation. Figure 6c displays the spectra for PAW-P and its interaction profile with the same hair types.

In Figure 6a, the UV–Vis spectrum of PAW-N prior to hair immersion exhibits a strong absorbance band in the 200–220 nm range, characteristic of NO₃⁻. After interaction with the hair samples, only slight reductions in absorbance are observed across all spectra, suggesting limited but detectable consumption of reactive species. The changes are subtle, indicating that most of the RONS remain in solution post-interaction, likely due to the short contact time or limited diffusion into the hair cuticle. Although slight variations are present, no substantial difference in spectral attenuation is observed among the different hair types, and thus no definitive correlation with porosity or surface area can be drawn from this specific spectral region alone.

The magnified spectral region presented in Figure 6b reveals distinct differences in absorbance between 250 and 400 nm following hair immersion. Sample 3 (straight hair) exhibits the most pronounced increase in absorbance near 300 nm, suggesting the formation of secondary oxidation byproducts, potentially nitro-compounds or aromatic degradation residues. This spectral shift may indicate localized oxidative reactions at the hair–liquid interface, reflecting heterogeneous chemical reactivity even within similar morphological types. Sample 4 (coily/kinky hair) also shows a moderate increase in this region, supporting the hypothesis that porous or chemically heterogeneous hair structures are more susceptible to oxidative interactions with nitrate-rich PAW-N. The overall trend in this zoomed-in region complements the SEM observations (Figure 3), where samples 3 and 4 showed more visible cuticle disruption, aligning with the presence of degradation products in the surrounding medium.

Figure 6c displays the UV–Vis spectra of peroxide-rich PAW-P before and after hair immersion, focusing on the spectral region between 190 and 280 nm. The initial PAW-P spectrum exhibits a broad absorption band centered around 200–220 nm, characteristic of hydrogen peroxide and related reactive oxygen species. After contact with hair, all samples show a discernible decrease in absorbance, indicating peroxide consumption during chemical interaction with the hair surface.

The most significant absorbance reductions are observed for Samples 1 and 4 (wavy and coily hair types), suggesting greater peroxide uptake, likely due to their higher porosity and larger surface area, which facilitate enhanced diffusion and redox reactions. In contrast, Samples 2 and 3 (straight hair) exhibit less pronounced changes, consistent with reduced surface reactivity and limited oxidative interaction.

These findings corroborate the SEM observations (Figure 4), where wavy and coily samples displayed greater morphological alterations, and support the hypothesis that hair type and structure play a critical role in mediating the chemical effects of PAW-P treatment. Unlike PAW-N spectra, no secondary peaks associated with oxidation byproducts are clearly resolved in this spectral window, which may indicate a cleaner oxidative pathway dominated by hydrogen peroxide with fewer aromatic or nitro-compound residues.

3.5. Mechanisms of Action of PAW-N and PAW-P on Different Hair Types

The distinct chemical profiles and oxidative capacities of PAW-N and PAW-P lead to markedly different effects on hair fibers, especially when considering intrinsic hair characteristics such as porosity, curl pattern, and cuticle density. As synthesized in

Table 5. Proposed mechanisms of action of PAW-N and PAW-P on different hair types, highlighting chemical interactions, structural effects, and hair integrity based on hair porosity and composition.

Feature/Effect	PAW-N (Nitrate-Rich)	PAW-P (Peroxide-Rich)	Hair Type Susceptibility
Chemical Composition	High NO3− (~500 mg/L), low pH (2.15), high ionic strength (6244 µS/cm)	High H2O2 (>25 mg/L), moderate pH (2.54), lower ionic strength (1926 µS/cm)	Coily (Type 4) and Wavy (Type 2) more vulnerable; straight (Type 1) more resistant
Oxidative Environment	Strong, driven by nitrate species and high conductivity	Moderate, driven by hydrogen peroxide with lower ionic stress	Coily and wavy hair show greater oxidative reactivity under PAW-N
Disulfide Bond Cleavage (1040 cm−1)	Extensive cleavage in wavy and one straight sample (1 and 2); retention or reformation in samples 3 and 4	Moderate and uniform increase in cysteic acid across all types	Higher susceptibility in porous types under PAW-N
Lipid Layer Degradation (2925–2850 cm−1)	Significant peroxidation, especially in coily and wavy hair	Partial degradation; lipid barrier largely preserved	Coily and wavy hair show greater lipid loss under PAW-N
Keratin Backbone Denaturation (Amide I/II)	Strong reduction in 1650 and 1550 cm−1 bands; major protein disruption	Mild reduction; keratin structure retained	More prominent in porous hair under PAW-N
Cuticle Integrity (SEM Analysis)	Pronounced fragmentation, lifting, and surface erosion	Mild lifting with overall structural preservation	Coily and wavy types exhibit more disruption under PAW-N
Moisture Loss (3400–3300 cm−1)	Marked dehydration and hydrogen bond disruption	Partial water loss; hydrogen bonding partially preserved	Greater moisture loss in porous hair under PAW-N
Lipid Oxidation (1730 cm−1)	Prominent oxidation products in coily and wavy hair	Limited oxidation; minor increase in lipid degradation bands	Lipid oxidation is mainly significant under PAW-N
Structural Rearrangement	Oxidative cleavage followed by partial reorganization (esp. in dense fibers)	Controlled oxidation promotes structural stabilization	Partial reformation in coily hair under PAW-P
Overall Hair Integrity	Compromised: increased porosity, cuticle damage, and protein loss	Preserved: surface-level oxidation with minimal core damage	PAW-P more appropriate for sensitive hair types

, PAW-N, dominated by high nitrate content (~500 mg/L), elevated ionic strength (6244 µS/cm), and low pH (2.15), creates an aggressive oxidative environment that promotes disulfide bond cleavage, lipid peroxidation, and significant protein denaturation. These effects are particularly detrimental to porous hair types, such as wavy (Type 2) and coily/kinky (Type 4) hair, which exhibited pronounced morphological damage and molecular degradation.

In contrast, PAW-P—characterized by high hydrogen peroxide concentration (>25 mg/L), lower ionic content (1926 µS/cm), and moderate acidity (pH 2.54)—induces milder oxidative effects. The FTIR and SEM data collectively show that PAW-P preserves cuticle integrity, reduces lipid loss, and maintains the keratin structure more effectively than PAW-N. All hair types benefited from PAW-P’s gentler profile, with straight hair (Type 1) being particularly resilient, showing minimal alterations in both morphological and molecular signatures. Coily hair, although generally more susceptible to oxidative stress, demonstrated partial structural reorganization under PAW-P, possibly due to its high surface area and dense cuticular architecture that moderate the penetration of reactive species.

To provide a clearer molecular basis for the observed effects, representative reaction pathways involving key biomolecules are outlined below:

(1)

Disulfide bond (cystine) oxidation in keratin [30]:

R–S–S–R′ + ROS → 2 R–SOH → R–SO₂H/R–SO₃H

(1)

(Stepwise oxidation leads to cleavage and conversion of disulfide bonds into sulfenic, sulfinic, and sulfonic acids, weakening the keratin matrix.)

(2)

Oxidative degradation of Vitamin A (retinol) [31]:

C₂₀H₃₀O + •OH → oxidized fragments + H₂O

(2)

(Breaks down lipid-soluble vitamins in the hair’s lipid layer, contributing to lipid barrier degradation.)

(3)

Nitration of tyrosine residues by peroxynitrite [32]:

ONOO⁻ + Tyr → 3-nitrotyrosine + OH⁻

(3)

(Nitrative stress alters amino acid side chains, potentially disrupting protein structure and stability.)

These simplified mechanisms are consistent with the spectroscopic signatures observed (e.g., changes at 1040 cm⁻¹, 2925–2850 cm⁻¹, 1650 cm⁻¹) and support the interpretation of oxidative and structural modifications induced by PAW. While not exhaustive, they provide a conceptual framework for understanding how different PAW formulations affect hair integrity at the molecular level.

This comparative mechanistic understanding underscores the importance of tailoring plasma-activated formulations to specific hair types and desired outcomes, balancing treatment efficacy with structural preservation.

4. Conclusions

This study investigated the physicochemical effects of PAW on human hair fibers with different morphologies, focusing on the comparative impact of two formulations: nitrate-rich PAW-N, generated Via a coaxial dielectric barrier discharge system, and peroxide-rich PAW-P, produced using a pin-to-water reactor. The results demonstrated that PAW-N, characterized by high nitrate concentration, low pH, and elevated ionic strength, induced significant oxidative damage throughout the hair structure, including disulfide bond cleavage, lipid peroxidation, protein denaturation, and pronounced cuticle fragmentation—effects particularly severe in porous hair types such as wavy (Type 2) and coily/kinky (Type 4). In contrast, PAW-P, defined by high hydrogen peroxide concentration and lower ionic content, resulted in milder oxidative interactions that preserved cuticle architecture, maintained keratin backbone integrity, and minimized moisture and lipid loss. Straight hair (Type 1), with its compact cuticle alignment and lower porosity, exhibited greater structural resilience under both treatments, especially when exposed to PAW-P. FTIR spectroscopy corroborated these findings through changes in cysteine oxidation (1034 cm⁻¹), hydrogen bonding (3400–3200 cm⁻¹), and lipid-associated vibrations (2925–2850 cm⁻¹), while SEM imaging confirmed surface-level degradation patterns. UV–Vis spectroscopy further revealed differential RONS depletion and byproduct formation, reinforcing the role of hair morphology in modulating chemical reactivity with PAW. Collectively, these findings underscore the importance of selecting PAW formulations according to hair type and treatment goals. While PAW-N may be suitable for applications requiring stronger oxidative action, its high reactivity poses a greater risk to fiber integrity. PAW-P, by contrast, offers a safer and more controlled alternative for cosmetic or routine use, particularly for porous and sensitive hair types. This work provides a mechanistic foundation for the rational development of plasma-based hair care strategies.