Effect of activated carbon microstructure and adsorption mechanism on the efficient removal of chlorophyll a and chlorophyll b from Andrographis paniculata extract

In order to reveal the effect of activated carbon (AC) properties on the adsorption of chlorophyll a (Chl a) and chlorophyll b (Chl b) in Andrographis paniculata extract, four commercial activated carbons were first tested and characterized. The results showed that activated carbon 1 (AC1) had the best surface area, pore structure and adsorption capacity. Therefore, adsorption isotherms, adsorption kinetics and adsorption mechanism were further carried out on AC1. The application of Langmuir model (R2 > 0.978) and Freundlich model (R2 > 0.977) indicated that the adsorption process of Chl a and Chl b on AC1 may be a complex adsorption process of single-layer and multilayer adsorption. The adsorption kinetics indicated that the pseudo-second-order kinetic model (R2 > 0.999) was dominant and was mainly chemisorption. The intra-particle diffusion model (R2 > 0.937) shows that the intra-particle diffusion is the rate-limiting step. The decrease of adsorption of AC1 to Chl a and Chl b due to the oxidation of acrylic acid proves the importance of π–π interaction.


Data analysis
Equilibrium adsorption capacity q e of Chl a and Chl b is calculated by the following mass balance equation: where q e /(mg g −1 ) is equilibrium adsorption capacity; C e /(mg L −1 ) is equilibrium mass concentration; C 0 /(mg L −1 ) is Chl a or Chl b concentration at zero time; W/(g) is the amount of AC; V/(L) is the volume of solution.
Langmuir model and Freundlich model were used to study adsorption isotherm: where q max /(mg g −1 ) is the maximum single-layer adsorption capacity; q t /(mg g −1 ) is the amount of adsorption at time t; K L /(mg L −1 ) is the adsorption constant.1/n is the heterogeneous factor, n represents the degree of isotherm deviation from linearity (1/n is between 0.1 and 1.0, indicating that the adsorption process is easy to proceed.) The thermodynamic parameter free energy (∆G 0 ) can be calculated by the following formula: The standard adsorption enthalpy (∆H 0 ) and the standard adsorption entropy (∆S 0 ) can be obtained by Van't Hoff 's formula: where R is the gas molar constant, R = 8.31 J mol K −1 ; T/(K) is the absolute temperature; Kc is the distribution coefficient, K C = C Ae C e , C Ae /(mg L −1 ) is the mass concentration of adsorbed substances and C e is the mass concentration of the remaining substance.
Adsorption kinetics of the AC was studied by the pseudo-first-order, pseudo-second-order and intra-particle diffusion kinetic models.
where k 1 /(min −1 ) and k 2 /(g mg −1 min −1 ) are the first order kinetic and the second order kinetic adsorption constants, and k p /(mg g −1 min −0.5 ) is the intra-particle diffusion rate constant, and C is constant which reflects the boundary layer effect.

Adsorption capacity of four activated carbons
Chl a and chl b differ in structure only in the additional groups on pyrrole ring II: the former is a methyl group, the latter is a formaldehyde group.Due to the extremely similar structure, if the same AC had a good adsorption capacity for chl a, its adsorption effect of chlorophyll b was also excellent.But the adsorption effect of different AC on the two adsorbed substances is greatly different.Four kinds of ACs adsorption effect is: AC1 > AC4 > AC2 > AC3 (Fig. 1), which shows that different manufacturers of AC for the same target adsorption capacity does have differences. (1) (2) t q e (pseudo-second-order model) www.nature.com/scientificreports/

Activated carbon characteristics
Figure 2 shows the SEM images of the four ACs magnification by 200, 1000 and 15,000 times respectively.All four ACs have wrinkles and depressions on their surfaces, which create favorable conditions for providing a large surface area.Under the 1000 magnification, the particle size of AC3 was observed to be significantly larger than  the other three, which indicated that the total specific surface area of AC3 may not be large, even if the surface of AC3 is honeycomb, which is thought to increase the total surface area of ACs.Under the 15,000 magnification, all four activated carbons have dense pores on their surfaces, and the mesoporous and microporous of ACs cannot be identified.
As shown in Fig. 3, the N 2 adsorption-desorption isotherms of the four ACs are similar to the type IV isotherms classified by the International Union of Purity and Applied Chemistry (IUPAC) 21 .All four N 2 adsorption-desorption isotherms showed H4 hysteresis loops composed of type I and type II isotherms with significantly adsorption capacity at the low end of P/P 0 , which suggested that the adsorption mechanism begins with the formation of single or multiple layers on the surface of AC, followed by pore condensation.Table 1 lists the calculated parameters of total specific surface area (S BET ), micropore specific surface area (S Mic ), total pore volume (V Tot ), micropore volume (V Mic ) and average pore diameter of the four ACs.
It has been reported that materials with large surface area and narrow pore distribution have more adsorption sites and higher adsorption energy, which has a significant influence on their adsorption behavior 22 .The S BET the four ACs from large to small is: AC1 (2525.68 m 2 g −1 ) > AC2 (2058.83m 2 g −1 ) > AC4 (1639.88m 2 g −1 ) > AC3 (948.23 m 2 g −1 ).The adsorption effect of four ACs was found to be AC1 > AC4 > AC2 > AC3.S Mic accounts for 70 ~ 90% of S BET .AC1 has the best adsorption effect with the largest S BET , while AC3 has the worst adsorption  www.nature.com/scientificreports/effect with the smallest S BET in four ACs; The S BET of AC2 is significantly greater than that of AC4, but its adsorption performance for Chl a and Chl b is inferior to AC4, which indicates that the S BET may play a major role in the adsorption process, but not a decisive role.The V Tot and pore distribution of the four ACs are different.From the experimental data, AC1 has the largest V Tot and the largest V Mic , AC3 has the smallest V Tot and the smallest V Mic , AC2 has the larger V Tot and V Mic than AC4, but the adsorption effect of AC2 is not as good as that of AC4.Therefore, the influence of V Tot and pore distribution on adsorption capacity cannot be clearly obtained.However, studies have shown that the most effective pore size of adsorbent is 1.76 ~ 6 times of the molecular size of adsorbent 23 .Chl a and Chl b differ in only one substituent, the distance between particular extreme atoms of Chl a molecule is about 1.256 nm in one direction and 1.104 nm in another one 24 .Therefore, the pore size suitable for Chl a and Chl b adsorption is about 2-8 nm.Saleem et al. 14 also showed that the size of dyes and humic acids (1.5 ~ 3.0 nm) was conducive to their adsorption in mesoporous pores.For adsorbents with molecular diameters of about 0.5 nm, microporous AC is usually sought as the primary adsorbent.The molecular sieve effect may greatly limit the adsorption, and the different pore size distribution may be one of the reasons for the different adsorption capacity of various ACs.The pore size distribution of four ACs is shown in Fig. 4.
Many chemical modifications have been used to increase the density of functional groups on the AC surface emphasizing the importance of functional groups interacting with polar substances in solution 25 .Figure 5 shows the FTIR spectra of four ACs and showed that the surfaces of all AC can act as donors and receptors for interaction, which is helpful in enhancing chlorophyll adsorption 20 .The peaks at 3319 cm −1 of AC3 and 3432 cm −1 of AC4 indicate the stretching vibration of -OH.The peak around 3000 cm −1 is probably the stretching vibration peak of the C-H bond on the benzene ring.The peak at around 1540 cm −1 is due to the aromatic C=C tensile vibration.The peak of 1000-1250 cm −1 may be the stretching vibration peak of C-O.The 1379 cm −1 peak of AC1 and 1345 cm −1 peak of AC4 indicate the possibility of C-N stretching vibration 21 .
The diffraction spectrum of amorphous matter is one with two peaks near 24° and 42°.Crystalline material will appear a set of sharp diffraction peaks, the sharper the peak, the higher the crystallinity 26 .Figure 6 shows that the four ACs all have the properties of crystalline substances, and the crystallinity in descending order is AC1 > AC4 > AC2 > AC3.

Adsorption isotherms
Langmuir model and Freundlich model are two kinds of adsorption isotherm models commonly used at present.According to the Langmuir model, the pollutants only covered the adsorbent in the form of a single molecular   layer, while in Freundlich model, the pollutants covered the adsorbent through multiple layers 26 .The Langmuir and Freundlich adsorption isotherms and parameters of Chl a and Chl b adsorbed on AC1 are shown in Fig. 7, Tables 2 and 3.
At different temperatures, the R 2 (ranging from 0.985 to 0.988) of Langmuir adsorption isotherms is relatively high; the R 2 (ranging from 0.996 to 0.998) of Freundlich adsorption isotherm models can well fit the adsorption processes, both together indicated that the adsorption process of Chl a and Chl b on AC1 may be a complex adsorption process of single-layer and multilayer adsorption.The equilibrium parameter R L of Langmuir model is close to 0 indicating that the adsorption process may be non-reversible adsorption.

Freundlich adsorption isotherm model parameters
K L (L mg −1 ) q e (mg g −1 ) R L (10 www.nature.com/scientificreports/In addition, with the increase of temperature, the K F of Freundlich model showed a upward trend, and the adsorption performance also increased slightly.The constant 1/n ranges from 0.1 to 1, indicating that the adsorption is easy to carry out 26 .
The thermodynamic parameters of adsorption of Chl a and Chl b by AC1 are shown in Table 4. ∆G 0 is all negative and the absolute value increases with the increase of temperature, indicating that the adsorption process is spontaneous, and rising temperature is conducive to the adsorption process.The positive value of ∆H 0 indicates that the adsorption of Chl a and Chl b by AC1 is an endothermic reaction.∆S 0 is positive, indicating that the adsorption process is an affinity adsorption, and the adsorption process is an entropy increasing process.
The AC1 used was stirred in a clean 90% ethanol solution for 2 h at 35 °C, 45 °C and 55 °C to determine whether the adsorption was reversible.It was found that only trace amounts of Chl a and Chl b were detected in the supernatant (Fig. 8), indicating that the adsorption was indeed irreversible adsorption.

Intra-particle diffusion model
The adsorption mechanism was studied by using the model of intra particle diffusion proposed by Weber and Morris 30 .As shown in Fig. 10, the Chl a and Chl b adsorbed quality (q t ) are linear to the square root curve of the contact time at all temperatures.The R 2 (ranging from 0.937 to 0.990) (Table 5) of intra-particle diffusion model is relatively high, which implied intra-particle diffusion may be a rate-limiting step 31 .The lines in the model do not pass through the origin, suggesting that other mechanisms of mass transfer are occurring at the same time as the diffusion within the particle.In addition, with the increase of temperature, the value of boundary thickness (C) increased, indicating that the increase of temperature in the adsorption process may lead to the transformation of adsorption mechanism from diffusion and transfer to boundary layer film.All the models studied in this paper are linear models.Compared with nonlinear models, linear models have the advantages of simple fitting, convenient fitting and fast fitting, but sometimes it will affect the accuracy of the model.For the sake of the intuitiveness of the data, we still use a linear model, which may be a weakness of this study.In future studies, it is necessary to further study the difference between linear and nonlinear fitting of AC to chl a and chl b adsorption models.

π-π interaction
Π-π interaction refers to the interaction between the π electron in the carbon adsorbent and the π electron in the aromatic ring of the adsorbates 32 .Both Chl a and Chl b contain four nitrogen; Nitrogen usually act as strong electron acceptors.AC is generally considered to have a hexagonal structure formed by covalent bonds with the large π-bond structural properties of aromatic rings.Therefore, strong π-π interaction between AC1 and Chl a and Chl b is possible.Acrylic oxidation experiments were used to determine the existence of π-π interaction.After adding oxygen groups to the aromatic ring structure of AC1, the adsorption rate decreased sharply (Fig. 11), which suggests that π-π interaction plays a vital role in the removal of Chl a and Chl b.The importance of π-π interaction has also been emphasized in the study of using AC to remove polycyclic aromatic hydrocarbons such as methylene green 33 , sulfamethoxazole 34 and naphthalene 35 .In addition, FTIR revealed the presence of nitrogen on the AC1 surface, and the presence of polycyclic aromatic hydrocarbons in Chl a and Chl b, which may provide another part of the π-π interaction.

Conclusion
The microstructures of four ACs were first characterized in order to reveal the internal factors for the difference in decolorization properties of Chl a and Chl b.The results show that the adsorption effect of AC on target adsorbent is affected by many factors.The S BET of the four ACs from large to small is: AC1 (2525.68 m 2 g −1 ) > AC2 (2058.83m 2 g −1 ) > AC4 (1639.88m 2 g −1 ) > AC3 (948.23 m 2 g −1 ), however, adsorption effect of four ACs is: AC1 > AC4 > AC2 > AC3.This suggests that the specific surface area may be dominant but not decisive in the adsorption process.All the four ACs N 2 adsorption-desorption isotherms show type IV isotherms with H4 hysteresis curves, which means that the four adsorbents have micropores and mesoporous.No strong correlation between pore volume, pore distribution and adsorption properties was observed in this study.FTIR shows that AC1 and AC4 contain nitrogen, which may enhance their adsorption of Chl a and Chl b.XRD results show that the four ACs have the properties of crystalline materials, and the crystallinity is AC1 > AC4 > AC2 > AC3.
AC1 has the best adsorption effect, so the adsorption isotherm, adsorption kinetics and adsorption mechanism were further studied on AC1.Langmuir model (R 2 > 0.978) and Freundlich model (R 2 > 0.977) indicated that the adsorption of Chl a and Chl b by AC1 may be a complex adsorption process of single layer adsorption and multilayer adsorption.The pseudo-second-order kinetic model (R 2 > 0.999) indicated that AC1 adsorption was a chemisorption.The practicability of intra-particle diffusion model (R 2 > 0.937) shows that intra-particle diffusion maybe a rate-limiting step.Oxidation experiments with acrylic acid show that π-π interaction is the key to the adsorption of Chl a and Chl b on AC1.
In conclusion, this study can not only provide reference for andrographolide manufacturers to screen AC for the removal of Chl a and Chl b, but also provide help for AC manufacturers to produce AC for the removal of Chl a and Chl b.

Figure 1 .
Figure 1.Adsorption capacity of four activated carbons for chlorophyll a and chlorophyll b at 35, 45 and 55 °C.

Figure 4 .
Figure 4. Pore size distribution of four activated carbons.

Figure 5 .
Figure 5.The FTIR spectra of four activated carbons.

Figure 6 .
Figure 6.X-ray diffraction patterns of four activated carbons.

Figure 7 .
Figure 7. Langmuir and Freundlich models of Chl a and Chl b of AC1.(a) Langmuir model of Chl a; (b) Freundlich model of Chl a; (c) Langmuir model of Chl b; (d) Freundlich models of Chl b.

Figure 9 .
Figure 9.The pseudo-first-order and pseudo-second-order model of Chl a and Chl b adsorbed on AC1.(a) pseudo-first-order of Chl a; (b) pseudo-second-order of Chl a; (c) pseudo-first-order of Chl b; (d) pseudosecond-order of Chl b.

Table 1 .
Structural parameters of four activated carbons.S BET Total specific surface area, S Mic Micropore specific surface area, V Tot Total pore volume, V Mic Micropore volume, S Ave Average pore size.

Table 2 .
Adsorption isotherm model parameters of Chl a on AC1.

Table 3 .
Adsorption isotherm model parameters of Chl b on AC1.

Table 4 .
Thermodynamic parameters for Chlorophyll a and chlorophyll b adsorption process by activated carbon 1.

Table 5 .
Pseudo-first order, pseudo-second order kinetics and intra-particle diffusion model parameters and correlation coefficients of Chl a and Chl b on AC1.