Novel ninhydrin-based deep eutectic solvents for amino acid detection

Journal Pre-proof Novel ninhydrin-based deep eutectic solvents for amino acid detection

Karzan A. Omar, Rahmat Sadeghi PII:

S0167-7322(19)37115-6

DOI:

https://doi.org/10.1016/j.molliq.2020.112644

Reference:

MOLLIQ 112644

To appear in:

Journal of Molecular Liquids

Received date:

25 December 2019

Revised date:

24 January 2020

Accepted date:

3 February 2020

Please cite this article as: K.A. Omar and R. Sadeghi, Novel ninhydrin-based deep eutectic solvents for amino acid detection, Journal of Molecular Liquids(2018), https://doi.org/ 10.1016/j.molliq.2020.112644

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2018 Published by Elsevier.

Journal Pre-proof 1

Novel Ninhydrin-based Deep Eutectic Solvents for Amino acid Detection

Karzan A. Omara,b, Rahmat Sadeghia a

b

Department of Chemistry, University of Kurdistan, Sanandaj, Iran

Department of Chemistry, Faculty of Science and Health, Koya University, Koya KOY45,

of

Kurdistan Region – F.R, Iraq

ro

Abstract

-p

The novel ninhydrin-based deep eutectic solvents (DESs) were synthesized from choline

re

chloride (ChCl) and tetrabutylammonium hydrogen sulfate (TBAHS) as hydrogen bond acceptors (HBAs) in a combination with ninhydrin as a hydrogen bond donor (HBD). In this

lP

work, the new HBA and HBD respectively including TBAHS and ninhydrin for the first time

na

were presented. The obtained deep eutectic solvents were further characterized at different temperatures with the results of density, sound velocity, viscosity, electrical conductivity,

Jo ur

refractive index, and surface tension data along with the 1H and 13C NMR, and FTIR analysis in order to confirm their purities, chemical structure, and occurrence interaction between the HBAs and HBD which formed the deep eutectic mixtures. The temperature dependences of their thermophysical properties were explored and correlated with simple linear models Jacobson and Arrhenius equation. The isothermal expansion (α), excess molar volume (VE), molar volumes (Vm), free volumes (fm), intermolecular free length (Lf), isentropic compressibility (β), molar conductivity (Λ), and molar refractions (Rm) values were calculated from the measured density, speed of sound, conductivity, and refractive index data. The synthesized DESs were applied as solvent and indicator reagent for quantitative and 

Corresponding author. [email protected]

Tel./fax.:

+988733624133.

E-mail

address:

[email protected]

and

Journal Pre-proof 2 qualitative determination of alanine. Also, the DESs displayed ability with high efficiency for replacement of organic solvents which are used in the amino acid detection processes.

Keyword: Deep eutectic solvents; Choline chloride; Tetrabutylammonium hydrogen sulfate; Ninhydrin; Amino acid detection.

1. Introductions

of

Ninhydrin is basically used to detect and analyze amino acids, proteins, peptides and other

ro

compounds which considered as ninhydrin-positive compounds [1]. Up to date, ninhydrin has

-p

been normally used as an essential element in the forensic and fingerprints detection [2]. As a

re

result of occurred reaction between amino acids and ninhydrin reagent, the deep blue or purple colors will be formed which known as Ruhemann's purple [3]. Moore and his co-

lP

worker modified ninhydrin reagent for the quantitative determination of the amino acid using

na

a spectrophotometer [4]. For this purpose, firstly the ninhydrin reagent are dissolved in the toxic organic solvents such as methyl cellosolve [5], isobutanol, ethanol, and acetone [6] and,

Jo ur

the buffer solution and other harmful corrosive acids such as HCl and H2SO4 are used for pH adjustment of amino acid solutions. Since 2003, the deep eutectic solvents (DESs) have become the most promising green solvents for volatile organic solvent replacement. DESs are synthesized from the combination of a hydrogen bond acceptor (HBA) with a hydrogen bond donor (HBD). Several researchers reported replacement of organic solvents by using DESs. In this regard, Gore and co-workers [7] presented the role of using tartaric acid-urea DES as an efficient solvent for high yield dihydropyrimidinones productions. Azizi et al. [8] reported the reline solvent for a chemoselective reduction in the presence of sodium borohydride. Also, in the polymerization filed, DESs benefitted as a proper solvent for the polymerization reactions [9]. Recently, most of the flavor extraction from plant materials carried out using DESs [10].

Journal Pre-proof 3 Even DESs became replaceable to its analogues ionic liquids due to their green solvent properties [11]. In this work, we present two novel ninhydrin-based DESs from the combination of choline chloride (ChCl) and tetrabutylammonium hydrogen sulfate (TBAHS) as HBAs with ninhydrin (Nin) as a HBD along with their physical properties. The synthesized DESs were applied as solvent and indicator reagent for quantitative and qualitative determination of alanine and the results showed that, the synthesized DESs displayed ability with high efficiency for replacement of organic solvents in the alanine detection processes. It

of

may be concluded that, DESs can successfully be applied for replacement of organic solvents

ro

which are used for the ninhydrin reagent preparation in order to qualitative and quantitative

re

-p

determination of amino acids by using spectrometry method.

2. Experimental

lP

2.1. Chemicals. TBAHS (≥ 99% w/w), Nin ((≥ 99% w/w) and Alanine ((≥ 99% w/w) were

na

obtained from Merck. ChCl (≥ 98% w/w) was obtained from Aldrich. TBAHS and ChCl were

in Fig. 1.

Jo ur

dried by heating &vacuum before use. Molecular structures of the used chemicals are shown

Fig. 1. Molecular structures of the chemicals used in this work

Journal Pre-proof 4 2.1. Synthesis of DESs. In this work, two DESs were prepared based on ChCl and TBAHS as HBA, and Nin as HBD in a 1:0.75 molar ratio (1 ChCl / TBAHS : 0.75 Nin). The digital balance (Sartorius CP225D) with an uncertainty of ±0.1 mg was used for weighting HBAs and HBD components. The mixtures of HBAs and HBD were heated at 100 °C and mixed by a magnetic stirrer at 300 rpm until the stable homogenous liquids were formed. The DES1 (ChCl-Nin) was found to have red color and the color of DES2 (TBAHS-Nin) changed from

of

green at 100 °C to light yellow at room temperature due to change in pH of TBAHS by temperature [12] (Fig. S1 of supplementary material). The synthesized DESs were kept in the

ro

tight bottles in desiccators for 50 days to prevent absorbing moisture and ensure their stability

-p

at room temperature. After this sufficient time their physical properties and applications were

re

studied. The DESs were synthesized under the drying conditions and were kept in the tight

lP

bottles in desiccators to preventing absorbing air moisture in order to avoid the effect of the

na

moisture on the DESs physical properties and their application.

2.2. Amino acid detection method. DES1 and DES2 were used for the qualitative and

Jo ur

quantitative determination of alanine. For this purpose, one drop of DES1 was added to 10 ml of a stock aqueous solution of alanine (with no further addition of an alkaline or acidic agent) and then blue color was appeared immediately. In the case of DES2, one drop of DES2 was added to 10 ml of a stock aqueous solution of alanine (containing 0.1M NaOH) and after 15 minutes the solution became blue color at room temperature (Fig. S2 of supplementary material). The same behavior was also observed at 100 °C which indicates that no change in the chemical structure of DES2 occurred at 100 °C. As can be seen from Fig. S3 of supplementary material, the blue color intensity of the solutions depends on the concentration of alanine. For the quantitative determination, a set of standard solutions of alanine were

Journal Pre-proof 5 made at various concentrations and after addition of DES, their UV–VIS absorption spectra were taken at wavelengths of 570 nm (max).

2.3. Physical properties measurements. The density and sound velocity of the synthesized DESs were simultaneously measured using a densimeter (Anton Paar DSA 5000). In advance to do the measurements, the U-tube shaped glass sample cell of densimeter was cleaned very

of

well by ethanol and double-distilled water to remove undesirable particles and obtain accurate results. The trace of moisture was dried by air-blower and vacuum pump for 20 minutes.

ro

After that, the density and sound velocity of the samples were measured in the range of

-p

293.15 to 333.15 K at 5 K intervals with the accuracy of ±0.00005 g.cm−3 and ±0.005 m.s-1

re

respectively. The refractive index measurements of the studied DESs were carried out using a

lP

digital Abbemat automatic refractometer (Anton Par, model WR), with temperature control accuracy of ±0.03 K. Firstly, the refractometer prism was cleaned with ethanol and dried with

na

the help of cotton to prevent any contamination which may possibly affect the determined results. After that, the double-distilled water was used for refractometer calibration at constant

Jo ur

temperature and pressure. The refractive index values of the DESs were measured three times at each temperature in the range of 293.15 to 333.15 K with a regular interval of 5 K at wavelength of 589.3 nm with an accuracy of ±4×10-5. Krüss K100 tensiometer was used for surface tension measurements of the studied DESs using the Du Noüy ring method [13]. Before starting measurement, the platinum–iridium ring was cleaned by ethanol and doubledistilled water and dried by flaming on the Bunsen burner. Double-distilled water was used for the calibration of tensiometer. Later, the surface tension measurements were carried out for the studied DESs at 303.15–333.15 K at 5 K intervals with replicate three times for each measurement. The uncertainty of the temperature and surface tension was around of ±0.1 K and ±0.2 mN/m, respectively. Brookfield Dial Viscometer was used for viscosity

Journal Pre-proof 6 measurements of the studied DESs at different temperatures (323.15 to 343.15 K with heating rate 1K per minute) and 12 RPM revolution using a spindle 64 with an adapter. All measurements were performed three times for each DESs at each temperature. Electrical conductivities of the synthesized DESs were determined using the Metrohm 712 conductivity meter, calibrated with 0.01 M of potassium chloride solution. To prevent water absorption, the glass vial which contains DESs and an electrode was very well sealed by paraflim. To control temperature, the glass vial was put into a water bath and connected to a thermostat.

of

The heating rate of the DESs samples was 1K per minute. Differential Scanning Calorimetery

ro

(DSC) (Model, DSC 205 F1, and Phoenix) was used for measuring the DESs freezing points.

-p

The instrument was equipped with a refrigeration system and auto-sampler and has the ability

re

to lower cooling limit 155.15 K. The nitrogen gas was used to clean the system with a flow rate of 50 ml/min. The indium metal was used for the calibration and baseline correction with

lP

an uncertainty smaller than 0.01. UV-Vis spectra were recorded using a SPEKOL 2000

na

double beam spectrophotometer (Analytik Jena), which has a slit width in a spectral range of 190 - 1100 nm for amino acid detection. Fourier transform infrared spectroscopy (FTIR)

Jo ur

analysis was recorded using Nicolet Impact 410 spectrometers. The absorption bands were measured in the range of 4000–500 cm-1 using KBr pellet method. 1H and

13

C NMR

measurements for the DESs were carried out at room temperature using Bruker AVANCE 400 spectrometer, 400 MHz. The synthesized DESs were prepared on 5 mm NMR tubes using almost 30 mg of DESs and then adding 0.5 ml of deuterium oxide (D2O) as solvent.

3. Results and discussion 3.1.

Freezing point. Fig. 2 shows the freezing point of mixtures of ChCl, TBAHS, and

Nin. A eutectic occurs at a ChCl and TBAHS to a Nin ratio of 1:0.75. The freezing point of the eutectic mixtures of DES1 and DES2 are 205 and 243 K, which are considerably lower

Journal Pre-proof 7 than that of their pure components (melting point of ChCl, TBAHS and Nin are 575, 446 and 523 K, respectively). The high freezing point property depression of the synthesized DESs must be raised from an interaction between hydrogen bond acceptor ions and Nin molecules. In fact, after forming DESs, all prepared molar ratio of HBA to HBD (1:0.25, 1:0.5, 1:0.75, 1:2, 1:1, and 2:1) were cooled down to room temperature and kept in the desiccator for more than 50 days. Most of the molar ratios were unstable and became solid during 24 hours,

of

except the molar ratio of 1:0.75 which was stable at room temperature. 600

ro

550

-p

500

re

400 350

lP

Tf / K

450

300

200 150

20

Jo ur

0

na

250

40

60

80

100

mole % Ninhydrin

Fig. 2. Freezing point of ○, DES1; ●, DES2 mixtures as a function of composition.

3.2. Density. Densities of the synthesized DESs were measured at different temperatures and the experimental data are reported in Table S1 of the supplementary material. The results show that, the densities of the DESs linearly decreased with increasing temperature and the density of DES based on the Nin/TBAHS is less than that of the Nin/ChCl DES. In fact, due to alkyl chain length of TBAHS which causes to increases the free volume, the density of DES2 is smaller than DES1 [14, 15]. The densities of the synthesized DESs were fitted to the following linear equation:

Journal Pre-proof 8 (1)

ρ = a + bT

where T is the absolute temperature, a and b are adjustable parameters that depend on the type of the studied DES. The values of the parameters a and b for two synthesized DESs along with their values of coefficient of determination, R2, average absolute relative deviation, ARD, and root mean square error, RMSE, were calculated and summarized in Table 1. The

of

low values and coefficient of determination indicated proper fit to the model.

Table 1. The values of the parameters a and b for two synthesized DESs along with R2, ARD

DES1

1.4283

-5.65

DES2

1.3072

-5.70

𝑒𝑥𝑝

a

𝐴𝑅𝐷 =

𝑐𝑎𝑙 1 𝑛 |𝜌𝑖 −𝜌𝑖 ∑𝑖=1 𝑒𝑥𝑝 𝑛 𝜌𝑖

| b

, 𝑅𝑀𝑆𝐸 =

R2

100ARDa

10-4.RMSEb

0.9992

0.086

3.94

0.097

3.15

-p

104b

re

a

lP

DES

ro

and RMSE

0.9997 0.5

𝑒𝑥𝑝 2

(𝜌𝑐𝑎𝑙 −𝜌 (∑𝑛𝑖=1 𝑖 𝑛−2𝑖

)

)

. n is number of experimental points, cal

na

and exp stand for the calculated and experimental density data, respectively.

Jo ur

The isobaric thermal expansion coefficient, α, of the DESs provides interesting information about expanding the DESs with temperature at a constant pressure. It can be calculated from the following equation: 1 𝜕𝜌

𝑏

𝛼 = − 𝜌 (𝜕𝑇) = − 𝜌 𝑃

(2)

where P is the pressure, and b the constant given in Table 1. The values of α for the DESs calculated from the fitting parameters given in Table 1 are depicted in Fig. 3 and reported in Table S2. It shows that the DESs expand with rising temperature and this expansion increases by increasing temperature. Furthermore, because of the larger free volumes, the values of α for DES2 are larger than DES1. The molar volumes, Vm, for both DESs were calculated from the experimental density data according to following equation:

Journal Pre-proof 9 𝑉𝑚 =

𝑥𝐻𝐵𝐴 𝑀𝐻𝐵𝐴 +𝑥𝐻𝐵𝐷 𝑀𝐻𝐵𝐷

(3)

𝜌

where x and M are the mole ratio and molar mass, respectively. The molar volumes of the DESs plotted with the variation of temperature have been shown in Fig. S4 (Table S3) of the supplementary material. As expected, the molar volumes of DESs slowly increase with increasing temperature. Likewise, from the measured densities data, the excess molar volumes, VE, were calculated by the following equation. 𝑥𝐻𝐵𝐴 𝑀𝐻𝐵𝐴 +𝑥𝐻𝐵𝐷 𝑀𝐻𝐵𝐷 𝜌

−

𝑥𝐻𝐵𝐴 𝑀𝐻𝐵𝐴 ∗ 𝜌𝐻𝐵𝐴

−

𝑥𝐻𝐵𝐷 𝑀𝐻𝐵𝐷

(4)

∗ 𝜌𝐻𝐵𝐷

of

𝑉𝐸 =

ro

where superscript * stands for the pure state of HBD or HBA. The calculated values of VE are

-p

given in Table S4 and shown in Fig. 3. 5.2

-38

lP na

4.6

4.4 280

Jo ur

104a / K-1

4.8

300

T/K

320

VE / cm3.mol-1

re

-39

5

-40 -41 -42 -43 -44

340

280

300

320

340

T/K

Fig. 3. Isobaric thermal expansion, α, and excess molar volume, VE, values of the synthesized DESs at different temperatures, T,: ○, DES1; ●, DES2.

It can be observed that the VE for both DESs have negative values due to the strong chemical interactions between HBAs and HBD molecules of the DESs system [16]. Furthermore, this figure shows that for both DESs the magnitude of the VE values decreases by increasing temperature. This may be related to this fact that the chemical interaction between HBA and HBD weakens by increasing temperature. At temperatures lower than about 305 K, the VE values for DES2 are more negative than DES1. However at temperature higher than 305 K,

Journal 10 Pre-proof the VE values for DES2 are less negative than DES1. In the other words, the slope for the plot of VE against T for DES2 are larger than DES1 which indicates that, the weakening of HBDHBA interaction by temperature for DES2 is larger than that for DES1.

3.3. Sound velocity The measured sound velocity data of both DESs as a function of temperature have been listed in Table S5 and their variations with temperature have been plotted in Fig. 4. The inverse

of

relation was observed between the sound velocity and temperature so that as temperature

ro

increases the values of sound velocity decrease. The traveling of sound waves through the

-p

medium depends on the medium densely. By increasing temperature, the DESs molecules

re

gain kinetic energy and move apart from each other, which causes to enhance free volume and reduce densities. This can be an explanation for slow traveling of sound waves in the less

lP

dense medium. For this reason, the molecules of DESs takes more time to travel through a

na

less dense liquid medium under high temperature in comparison to the denser medium under the low temperature [17]. The effect of temperature on the retarding of sound velocity in the

Jo ur

liquid medium can be verified by Jacobson [18] and Newton-Laplace equation [19, 20]. The Jacobson equation, which can be used for determining the intermolecular free length at different temperature, has the following form: 𝐾

𝐿𝑓 = 𝑢𝜌0.5

(5)

where K is the Jacobson's constant, ρ is density in g·cm-3 and u is sound velocity in m·s-1. The calculated values of (Lf) plotted as function of temperature has been given in Fig. S5 of supplementary material. That is to say, the free space has a direct relationship with temperature so that, raising temperature is caused to lower wave travelling in a medium due to creating more free space.

Journal 11 Pre-proof 2600

400

300

2200

β / TPa-1

u / m.s-1

2400

2000

200

100

1800

0

1600 280

300

320

290

340

310

330

350

T/K

of

T/K

ro

Fig. 4. Sound velocity, u, and isentropic compressibility, β, values of the synthesized DESs at different

-p

temperatures: ○, DES1; ●, DES2.

re

The isentropic compressibility, β, which is an important parameter and provides information

lP

about the existence of free space in the liquid materials at various temperatures, can be determined from the Newton-Laplace equation: 1

na

𝛽 = 𝜌𝑢2

(6)

Jo ur

As shown in Fig. 4 (and Table S2 of supplementary material), the isentropic compressibility increases with increasing temperature. At higher temperatures, because of the more free space, the DESs become more compressible. The similar behavior has also been observed for organic solvents, ILs and water soluble polymers [21]. On the other hand, the compressibility of pure water decreases with temperature to a minimum β value near 337.15 K and then increases gradually [21].

3.4. Conductivity Conductivities of the synthesized DESs were measured in the temperature range of (293.15 to 333.15) K under atmospheric pressure. Ionic conductivity strongly depends on the viscosity,

Journal 12 Pre-proof temperature, and the alkyl chain length [22]. The electrical specific conductivity, , of the DESs are reported in Tables S6 and plotted as a function of temperature in Fig. 5. As can be seen, the conductivities of both DESs increase with increasing temperature and also DES2 because of its longer alkyl chain length has a smaller conductivity than DES1. The molar conductivity,m, of DESs can be calculated by the following equation: Λ𝑚 =

𝜅𝑀

(7)

𝜌

of

From Fig. 5 (Table S6), it can be seen that molar conductivity has a similar behavior with the

ro

specific conductivity.

The variation of specific conductivity with temperature was fitted to the Arrhenius equation: 𝐸

-p

𝜅 = 𝜅 ° 𝑒𝑥𝑝 (− 𝑅𝑇𝑎 )

(8)

re

where º is pre-exponential factor, 𝐸a is the activation energy of electrical conduction and R is

lP

the gas constant. The values of the adjustable parameters º and 𝐸a for both DESs are listed in

na

Table 2. 200

 / μs.cm-1

150

100

30000

(b)

m / μs.cm-1.mol-1

Jo ur

(a)

50

0

20000

10000

0 280

300

320

T/K

340

290

300

310

320

330

340

T/K

Fig. 5. a) Specific conductivity and b) molar conductivity of the synthesized DESs at different temperatures, ○, DES1; ●, DES2; ─, calculated by Eq. (8).

Journal 13 Pre-proof Table 2. The values of the adjustable parameters º and 𝐸a for the synthesized DESs along with R2

DES

1015º / μs.cm-1

105𝐸a / J.mol-1

R2

DES1

0.0024

-0.647

0.9987

DES2

125.3

-1.010

0.9987

3.5. Viscosity The viscosities of the synthesized DESs were measured in the temperature range of (323.15 to

of

343.15) K under atmospheric pressure and the experimental viscosity data have been given in

ro

Table S7 of the supplementary material. At temperatures below 323.15 K, both DESs

-p

demonstrate very high viscosities. As shown in Fig. 6, at high temperatures, the DESs exhibit

re

a slight increase in their viscosities with decreasing temperature, bending up at low temperatures. DES2 because of its longer alkyl chain length and higher molar mass has a

lP

larger viscosity than DES1. The presence of a massive hydrogen network and other

na

interactions, resulting in high viscosity and low ionic mobility in the small void volume within the DESs [22-24]. The DES2 has a highest viscosity of 48500 mPa.s at 323.15 K,

Jo ur

while DES1 has a lower viscosity of 30500 mPa.s at 323.15 K. The variation of viscosity, η, with temperature was fitted to the Arrhenius equation: 𝐸𝜂

𝜂 = 𝜂° 𝑒𝑥𝑝 (𝑅𝑇)

(9)

where Eη is the activation energy for momentum transfer (J.mol-1), and ηº is pre-exponential factor. Table 3 lists the values of Eη, ηº and the regression coefficients for the DESs.

Table 3. The values of Eη and ηº for two synthesized DESs along with R2. DES

10-11ηº / mPa.s

104Eη / J.mol-1

R2

DES1

0.527

9.750

0.9997

DES2

1.225

9.646

0.9997

Journal 14 Pre-proof 50000

η / mPa.s

40000 30000 20000 10000 0 325

330

335

345

ro

T/K

340

of

320

Fig. 6. Viscosity of the synthesized DESs at different temperatures, ○, DES1; ●, DES2; ─, calculated

re

-p

by Eq. (9).

3.6. Refractive index

lP

The refractive index values of the synthesized DESs measured in the temperature range of

na

(293.15 to 333.15) K at atmospheric pressure are summarized in Table S8 of the supplementary material. The variation of refractive index with temperature is shown in Fig. 7.

Jo ur

The observed results of the DESs refractive index revealed that the value of nD decreases with increasing temperature. This behavior is similar to the DESs densities. At each temperature the value of refractive index of DES2 is smaller than that of DES1 in results of the effect of long alkyl chain length [25]. Moreover, the refractive index values can be related to the electronic polarizability of molecules. Therefore, the high values for refractive index of the DESs can be attributed to the existence of a polarizable aromatic ring [26]. The variation of refractive index, nD, with temperature was fitted to the following equation: nD = a + bT

(10)

where a and b are constants parameters and Table 4 displays their values along with the values of R2 for the synthesized DESs.

Journal 15 Pre-proof Table 4. The values of adjustable parameters of Eq. (10) (a and b) for two synthesized DESs along with R2. DES

a

10-4b

R2

DES1

1.57

-3.0

0.9999

DES2

1.514

-2.7

0.9999

As can be seen, good agreement was observed between the calculated (by Eq. (10)) and

of

experimental values of nD with regression coefficient values of > 0.99. From the measured

ro

refractive index data, the values of molar refractivity, Rm, and free molar volume, Fm, were calculated by the following equations: 2 𝑀(𝑛𝐷 −1)

-p

𝑅𝑚 =

2 +1) 𝜌(𝑛𝐷

(12)

re

Fm = Rm - Vm

(11)

lP

According to the Lorenz-Lorentz approximation, the molar refractive values represent a hard

na

core molecular volume. Basically, the assumption made in the liquid state in their molecular structure, there is a free volume and not occupied known as free molar volume [27, 28]. The

1.59

(a)

Jo ur

plot of Rm and Fm against temperature are shown in Fig. 7.

800

(b)

700

nD

Rm / cm3.mol-1

1.54

1.49

600 500 400 300 200

1.44 280

300T / K

320

340

280

300

T/K

320

340

Journal 16 Pre-proof 450

(c) 400

Fm / cm3.mol-1

350 300 250

of

200 150 300

310

T/K

320

330

340

ro

290

-p

Fig. 7. a) Refractive index, b) molar refractivity, and c) free molar volume of the synthesized DESs at

re

different temperatures, ○, DES1; ●, DES2; ─, calculated by Eq. (10).

lP

3.7. Surface tension

The surface tension measurements of the DESs were carried out at ambient pressure and

na

temperature ranges of (308.15 to 333.15) K and the measured data are listed in Table S9 of the supplementary material. The change in surface tension of the DESs with temperature has

Jo ur

been plotted in Fig. 8. The surface tensions of the DESs decrease with temperature in the results of disrupting of hydrogen bond network. Also, the long alkyl chain length reduces the surface tension of DESs so that DES2 has less surface tension than DES1, due to its long alkyl chain length [29]. The variation of surface tension, , with temperature was fitted to the following equation:

 = a + bT

(13)

where a and b are constants parameters and Table 5 displays their values along with the values of R2 for the synthesized DESs.

Journal 17 Pre-proof 80

 / mN.m-1

70 60 50 40 30 20 310

315

320

325

T/K

330

of

305

335

ro

Fig. 8. Surface tension of the synthesized DESs at different temperatures, ○, DES1; ●, DES2; ─,

re

-p

calculated by Eq. (13).

Table 5. Surface tension fitting parameters and regression coefficients obtained from Eq. (13).

b

R2

96.739

-0.107

0.9999

99.999

-0.203

0.9999

a

DES1 DES2

Jo ur

na

lP

DES

3.8. 1H and 13C NMR

The chemical structure and purity of the synthesized DESs were confirmed by 1H and

13

C

NMR spectroscopy. To provide more comprehensible information about the purity and the nature of interactions, the 1H and 13C NMR spectra of both DESs were recorded (Fig. 9). The DESs signals compared with each individual component and difference in their chemical shift observed. The signals for formation of hydrogen bond can be observed at 7.547 and 7.707 ppm for DES1 and DES2, respectively, which indicate the strong interactions between HBAs (ChCl and TBAHS) with Nin as a HBD. Furthermore, the H signals at 7.547 and 7.707 ppm were ascribed to those in ninhydrin. It was found that the H signals in DESs were shifted to

Journal 18 Pre-proof down-field shift in comparing to two signals of a pure component (Table S10 of the supplementary material). Signals at δ(ppm)=3.146 (9H, choline chloride CH3), δ(ppm)=3.477 (2H, choline chloride CH2), δ(ppm)=3.954 (2H, choline chloride CH2), (ppm)=7.547 (H, ninhydrin OH), δ(ppm)=7.636 (H, ninhydrin CH), δ(ppm)=0.647 (3H, TBAHS CH3), δ(ppm)=1.062 (2H, TBAHS CH2), δ(ppm)=1.320 (2H, TBAHS CH2), δ(ppm)=2.883 (2H, TBAHS CH2), δ(ppm)=7.707 (H, ninhydrin OH), and δ(ppm)=7.785 (H, ninhydrin CH) can be demonstrated as characteristic 1H-NMR signals of both DESs protons were deshielding to

of

hydrogen atoms, which indicate the reduction in the electron density of hydrogen atoms,

13

C-NMR spectrum which shown in Figs. S6 and S7 of the supplementary

-p

confirmed by

ro

resulting in the down-filed shift of the chemical shifts [30, 31]. The purity of DESs was

re

material. Signals at δ = 54.16-87.46, 87.46-197.24 and 12.96-57.80 are related to aliphatic carbon of choline chloride, carbon of ninhydrin and aliphatic carbon of TBAHS (CH3),

lP

respectively. As can be observed, no impurities could be found in 1HNMR and

Jo ur

na

spectra of both DESs.

(a)

(b)

Fig. 9. 1H NMR spectra of DES1 (a) and DES2 (b) in D2O solvent.

13

C-NMR

Journal 19 Pre-proof 3.9. FT-IR Fig. 10 shows the comparison between the FTIR spectra of ChCl, TBAHS, Nin, DES1 and

Jo ur

na

lP

re

-p

ro

of

DES2.

Journal 20 Pre-proof

a-[ChCl]

3299.87

2970.14

-p

ro

of

b-[TBAHS]

d-[DES1]

Jo ur

na

lP

re

c-[Nin]

3274.96

3449.53

e-[DES2]

Journal 21 Pre-proof Fig. 10. Infrared spectroscopy of a) ChCl, b) TBAHS, c) Nin, d) DES1, and e) DES2.

The FT-IR spectrum of DES1 and DES2 show the frequency combination of ChCl, TBAHS and Nin. A sharp peak at 1383.58 cm−1 in Fig. 10d corresponds to the C-O bond stretching of ChCl, and 1428.95 cm−1 in Fig. 10e corresponds to the S=O bond stretching of TBAHS. The peaks at 3299.87 cm−1 (Fig 10a, ChCl), 3274.96 cm−1 (Fig. 10c, Nin), 3449.53 cm−1 (Fig. 10d, DES1) and 3450.83 cm−1 (Fig. 10e, DES2) were assigned to stretching vibration of the O-H

of

group. As can be seen, a shift in the OH stretching vibration occurred when the DESs were

ro

formed. In the other words, because of the presence of hydrogen-bonding interaction between the HBD and HBAs, the broad peaks of the hydroxyl groups of DESs shift to the higher

-p

frequency [32, 33]. In fact, transfer of electron cloud of oxygen atom to the hydrogen bond

re

leads to decreasing in the force constant [34] and therefore, the shift of the OH stretching

lP

vibration to the higher frequency can be attributed to forming the hydrogen bond between

Jo ur

4. Applications

na

HBAs and HBD when the DESs are generated.

4.1. Organic solvent replacement

For centuries, the ninhydrin compound has been used in medical, food, agricultural, and forensic science for detecting fingerprint [35]. Also, it is applicable for detecting amino acids [36] as well as for the quantitative determination of several amino acids by using spectrometric method [5, 6, 37]. Since 1910, the ninhydrin reagent was being dissolved and prepared in organic solvents including acetone, ethanol, isobutanol, and methyl cellosolve. All these organic solvents are toxic, volatile, and neither environmental friendly nor safe. In this work, we present two new deep eutectic solvents for replacing these organic solvents, which are safe, nonvolatile, biocompatible and environmentally friendly. During the detection of alanine, one drop of DES1 was used as solvent and indicator reagent for reacting with

Journal 22 Pre-proof alanine with no further adjustment. But, for the DES2 the pH of stock solution was adjusted using sodium hydroxide to stabilize formed color due to the acidity of DES2. By this way, we are able to replace toxic organic solvents by the novel DESs green solvents. In fact, as one drop of the DESs is added to pure alanine or an aqueous alanine solutions the blue color is appeared.

4.2. UV-Visible spectroscopy calibration curve for alanine determination

of

As shown in Fig. 11, the proposed method for alanine determination exhibits a maximum

ro

absorption at 570 nm. Under such condition, the calibration curve of the absorbance against

-p

the alanine concentration was found linear in the concentration range 0.1-0.8 mg/mL for

re

DES1 and 0.01-0.06 mg/mL for DES2. The determined absorbance plotted versus

1.4

na

1 0.8 0.4 0.2 0 470

Jo ur

0.6

570

4 3 2 1 0 0

670

Wavelength (λmax)

0.5 Concentration (mg/mL)

(b)

(a) 2.5 Optical density

Optical density

1.2

5

Optical density

lP

concentration as shown in Fig. 11.

2 1.5 1 0.5 0 0

0.02

0.04

Concentration (mg/mL)

©

0.06

Journal 23 Pre-proof Fig. 11. λmax determination (a) of alanine stock solution in the presence of both DESs, calibration curve of alanine in aqueous solution using DES1 (b) and DES2 (c) at 570 nm. The linear regression of absorbance on concentration gave equation y = 4.7545x + 0.1555 with a correlation of 0.9967 for DES1 and y = 41.491x + 0.1228 with a correlation of 0.9978 for DES2.

5. Conclusions

of

Two novel ninhydrin-based deep eutectic solvents as novel green solvents were presented and

ro

studied in this work. These new synthesized DESs, which presented high depression freezing

-p

points, were composed of ChCl and TBAHS as hydrogen bond acceptors and ninhydrin as

re

hydrogen bond donors and their physical properties such as density, speed of sound, conductivity, refractive index, surface tension, and viscosity were determined at different

lP

temperatures and atmospheric pressure. From these experimental physical properties, the

na

values of isothermal expansion, excess molar volume, molar volume, isentropic compressibility, free volume, intermolecular free length, molar conductivity, and molar

Jo ur

refraction were determined. The values of isothermal expansion, isentropic compressibility, free volume and viscosity for DES2 are larger than DES1. However the values of conductivity and surface tension for DES1 are larger than DES2. There are two factors that affect the physical properties of the synthesized DESs. (1) Power of hydrogen bond interaction between HBA and HBD and (2) alkyl chain length of HBA (which caused the difference between the molar volume of HBA and HBD). The higher freezing point depression of DES1 than DES2 shows that the hydrogen bond interaction between ChCl and Nin is stronger than that between TBAHS and Nin. These two factors have opposite effect on the VE values so that at temperatures lower than about 305 K, the later factor is predominant and therefore the VE values for DES2 are more negative than DES1. However at temperature

Journal 24 Pre-proof higher than 305 K, the first factor is predominant and therefore the VE values for DES2 are less negative than DES1. In the case of the other thermodynamic properties, these two factors have parallel effect and therefore there is no any crossing of the plots of thermodynamic properties against temperature. Finally, the new DESs were applied as an organic solvent replacement for qualitative and quantitative determination of amino acid alanine by using spectrometric method.

of

References

-p

ro

[1] M. Friedman, Applications of the Ninhydrin Reaction for Analysis of Amino Acids, Peptides, and Proteins to Agricultural and Biomedical Sciences, Journal of Agricultural and Food Chemistry 52 (2004) 385-406. [2] I. S. G. Drochioiu, R. Gradinaru, G. Zbancioc, I. Mangalagiu, Ninhydrin-based Forensic Investigations: II. Cyanide Analytical Toxicology. International Journal of Criminal Investigation

re

1 (2011) 213-226.

Jo ur

na

lP

[3] S. Ruhemann, CXXXII.—Cyclic di- and tri-ketones, Journal of the Chemical Society, Transactions 97 (1910) 1438-1449. [4] S. Moore, W. H. Stein, A Modified Reagent for the Photometric Determination of Amino Acids and Related Compounds. Journal of Biological Chemistry 211 (1954) 907-913. [5] L. Fowden, The Quantitative Recovery and Colorimetric Estimation of Amino-Acids Separated by Paper Chromatography, Biochemical Journal 48 (1951) 327-333. [6] W. H. Fitzpatrick, Spectrophotometric Determination of Amino Acids by the Ninhydrin Reaction, Science 109 (1949) 469. [7] S. Gore, S. Baskaran, B. Koenig, Efficient Synthesis of 3,4-Dihydropyrimidin-2-ones in Low Melting Tartaric Acid–Urea Mixtures, Green Chemistry 13 (2011) 1009-1013. [8] N. Azizi, E. Batebi, Highly Efficient Deep Eutectic Solvent Catalyzed Ring Opening of Epoxides, Catalysis Science & Technology 2 (2012) 2445-2448. [9] A.V. Gómez, A. Biswas, C. C. Tadini, R. F. Furtado, C. R. Alves, H. N. Cheng, Use of Natural Deep Eutectic Solvents for Polymerization and Polymer Reactions, Journal of the Brazilian Chemical Society 30 (2019) 717-726. [10] C. G. González, N. R. Mustafa, E. G. Wilson, R. Verpoorte, Y. H. Choi, Application of Natural Deep Eutectic Solvents for the “Green” Extraction of Vanillin from Vanilla Pods, Flavour and Fragrance Journal 33 (2018) 91-96. [11] F. Merza, A. Fawzy, I. AlNashef, S. Al-Zuhair, H. Taher, Effectiveness of Using Deep Eutectic Solvents as an Alternative to Conventional Solvents in Enzymatic Biodiesel Production from Waste Oils. Energy Reports 4 (2018) 77-83. [12] D. A. MacFadyen, On the Mechanism of the Reaction of Ninhydrin with α-Amino Acids: I. Absorption Spectra of Ninhydrin and Certain Derivatives. Journal of Biological Chemistry 186 (1950) 1-29.

[13] P. L. du Noüy, An Interfacial Tensiometer for Universal Use. The Journal of General Physiology 7 (1925) 625-631.

Journal 25 Pre-proof

Jo ur

na

lP

re

-p

ro

of

[14] G. García, S. Aparicio, R. Ullah, M. Atilhan, Deep Eutectic Solvents: Physicochemical Properties and Gas Separation Applications, Energy & Fuels 29 (2015) 2616-2644. [15] M. G. Montalbán, C. L. Bolívar, F. G. Díaz Baños, G. Víllora, Effect of Temperature, Anion, and Alkyl Chain Length on the Density and Refractive Index of 1-Alkyl-3methylimidazolium-Based Ionic Liquids, Journal of Chemical & Engineering Data 60 (2015) 1986-1996. [16] R. S. Achsah, S. Shyam, N. Mayuri, R. Anantharaj, Thermodynamic Properties of Deep Eutectic Solvent and Ionic Liquid Mixtures at Temperatures from 293.15 K to 343.15 K. AIP Conference Proceedings 1951 (2018) 020010. [17] F. S. Mjalli, N. M. Abdel Jabbar, Acoustic Investigation of Choline Chloride Based Ionic Liquids Analogs. Fluid Phase Equilibria 381 (2014) 71-76. [18] B. Jacobson, Ultrasonic Velocity in Liquids and Liquid Mixtures. The Journal of Chemical Physics 20 (1952) 927-928. [19] G. Sharma, R. L. Gardas, A. Coronas, G. Venkatarathnam, Effect of Anion Chain Length on Physicochemical Properties of N,N-Dimethylethanolammonium Based Protic Ionic Liquids. Fluid Phase Equilibria 415 (2016) 1-7. [20] D. Singh, R. L. Gardas, Influence of Cation Size on the Ionicity, Fluidity, and Physiochemical Properties of 1,2,4-Triazolium Based Ionic Liquids. The Journal of Physical Chemistry B 120 (2016) 4834-4842. [21] R. Sadeghi, H. Shekaari, R. Hosseini, Effect of Alkyl Chain Length and Temperature on the Thermodynamic Properties of Ionic Liquids 1-Alkyl-3-methylimidazolium Bromide in Aqueous and Non-aqueous Solutions at Different Temperatures. The Journal of Chemical Thermodynamics 41 (2009) 273-289. [22] Q. Zhang, K. De Oliveira Vigier, S. Royer, F. Jerome, Deep Eutectic Solvents: Syntheses, Properties and Applications. Chemical Society Review 41 (2012) 7108-7146. [23] S. Sarmad, Y. Xie, J.-P. Mikkola, X. Ji, Screening of Deep Eutectic Solvents (DESs) as Green CO2 Sorbents: From Solubility to Viscosity. New Journal of Chemistry 41 (2017) 290-301. [24] M. K. AlOmar, M. Hayyan, M. A. Alsaadi, S. Akib, A. Hayyan, M. A. Hashim, Glycerol-Based Deep Eutectic Solvents: Physical Properties. Journal of Molecular Liquids 215 (2016) 98-103. [25] Z. Chen, M. Ludwig, G. G. Warr, R. Atkin, Effect of Cation Alkyl Chain Length on Surface Forces and Physical Properties in Deep Eutectic Solvents. Journal of Colloid and Interface Science 494 (2017) 373-379. [26] A. P. Abbott, D. Boothby, G. Capper, D. L. Davies, R. K. Rasheed, Deep Eutectic Solvents Formed between Choline Chloride and Carboxylic Acids:  Versatile Alternatives to Ionic Liquids. Journal of the American Chemical Society 126 (2004) 9142-9147. [27] G. Li, Y. Jiang, X. Liu, D. Deng, New Levulinic Acid-Based Deep Eutectic Solvents: Synthesis and Physicochemical Property Determination. Journal of Molecular Liquids 222 (2016) 201-207. [28] A. Basaiahgari, S. Panda, R.L. Gardas, Acoustic, Volumetric, Transport, Optical and Rheological Properties of Benzyltripropylammonium Based Deep Eutectic Solvents. Fluid Phase Equilibria 448 (2017) 41-49. [29] Y. Marcus, Properties of Deep Eutectic Solvents, Deep Eutectic Solvents, Springer International Publishing, Cham, 2019, pp. 45-110. [30] L. Hao, M. Wang, W. Shan, C. Deng, W. Ren, Z. Shi, H. Lü, L-Proline-Based Deep Eutectic Solvents (DESs) for Deep Catalytic Oxidative Desulfurization (ODS) of Diesel. Journal of Hazardous Materials 339 (2017) 216-222.

Journal 26 Pre-proof

Jo ur

na

lP

re

-p

ro

of

[31] C. Li, D. Li, S. Zou, Z. Li, J. Yin, A. Wang, Y. Cui, Z. Yao, Q. Zhao, Extraction Desulfurization Process of Fuels with Ammonium-Based Deep Eutectic Solvents. Green Chemistry 15 (2013) 2793-2799. [32] C. Florindo, F. S. Oliveira, L. P. N. Rebelo, A. M. Fernandes, I. M. Marrucho, Insights into the Synthesis and Properties of Deep Eutectic Solvents Based on Cholinium Chloride and Carboxylic Acids. ACS Sustainable Chemistry & Engineering 2 (2014) 2416-2425. [33] A. Abri, N. Babajani, A. M. Zonouz, H. Shekaari, Spectral and Thermophysical Properties of Some Novel Deep Eutectic Solvent Based on l-Menthol and Their Mixtures with Ethanol. Journal of Molecular Liquids 285 (2019) 477-487. [34] W. Guo, Y. Hou, W. Wu, S. Ren, S. Tian, K. N. Marsh, Separation of Phenol From Model Oils with Quaternary Ammonium Salts Via Forming Deep Eutectic Solvents. Green Chemistry 15 (2013) 226-229. [35] S. OdÉN, B. V. Hofsten, Detection of Fingerprints by the Ninhydrin Reaction. Nature 173 (1954) 449-450. [36] S. J. Sheng, J. J. Kraft, S. M. Schuster, A Specific Quantitative Colorimetric Assay for L-Asparagine. Analytical Biochemistry 211 (1993) 242-249. [37] A. M. Smith, A. H. Agiza, The Determination of Amino-acids Colorimetrically by the Ninhydrin Reaction. Analyst 76 (1951) 623-627.

Journal 27 Pre-proof Declaration of competing interests

☒ 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.

Jo ur

na

lP

re

-p

ro

of

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Journal 28 Pre-proof

Author Statement

Jo ur

na

lP

re

-p

ro

of

Karzan A. Omar: Conceptualization, Methodology, Data curation, Writing-Original draft preparation, Resources. Rahmat Sadeghi: Supervision, Writing- Reviewing and Editing,

Journal 29 Pre-proof

Jo ur

na

lP

re

-p

ro

of

Graphical abstract

Journal 30 Pre-proof Highlights Ninhydrin was paired with two hydrogen bond acceptors to form new DESs.



The physical characteristic of the Ninhydrin-based DESs were determined and fitted.



Present green solvent has a green potential for organic solvent replacement.



New DESs were applicable for qualitative and quantitative amino acid determination.

Jo ur

na

lP

re

-p

ro

of

