The confirmation test of the solid product was done using 0.01N NaOH and the confirmation of Ca2+linkage was done, When NaOH reacts with Ca²⁺ ions, it forms calcium hydroxide (Ca (OH)₂), which is sparingly soluble in water and forms a white precipitate. The reaction is represented as follows:
Ca²⁺ + 2NaOH ---Ca (OH)₂↓ + 2Na⁺
The formation of this white precipitate (Fig. 3) is a positive indication of the presence of calcium ions.
Identification of Ca2+ ion attachment through IR from K+ linked carrageenan.
Since calcium ions (Ca2+) do not have any identifiable IR absorption bands, identifying Ca2+ ion attachment using IR spectroscopy can be difficult. However, Ca2+ ions have distinctive IR absorption bands that is utilized to distinguish them when they bind with another compounds. The comparison of the IR of K+ and Ca2+ion attachment using IR spectroscopy have been done through some of the IR peaks given in the Fig. 4.
The peak observed at 1644 cm-1, corresponding to the O-H bending "deformation," provides insights into the presence of water bonded to the polymer. This peak has shifted to a higher infrared (IR) value and exhibits a higher intensity. These observations suggest that the K+ ions have been replaced, likely due to the smaller size of Ca2+ ions, which results in a stronger attraction towards water and sulfate ions. The O-S stretching frequency in the presence of Ca2+ ions is measured at 1288 cm-1 with an absorbance of 85%. In contrast, when K + ions are present, the O-S stretching frequency is observed at 1304 cm− 1 with an absorbance of 70%.[20] Additionally, this observation indicates the presence of a strong interaction and an accompanying decrease in bond length. This phenomenon results in a drop in the S-O frequency, indicating that the S-O–Ca2+ contacts are stronger compared to the S-O----K+ interaction. The observed displacement of the peak and its corresponding intensity provides confirmation of the substitution of K+ ions with Ca2+ ions in the carrageenan, while leaving the fingerprint region unaffected (721cm− 1, 1456 − 1300 cm− 1 two intense peaks of SO2, 1280–1300 cm− 1of S-O bond.) [21–23]
Mass spectroscopy.
The mass spectra provide compelling evidence for the substitution of K+ ions with Ca2+ ions in the iota carrageenan. (Fig. 5) The presence of the fragment peak [136]+(477 − 341) provides confirmation of the existence of the [SO4Ca]+ fragment. Similarly, the presence of the [154]+ (221 − 77) fragment verifies the presence of [SO4Ca2+. H2O]+, indicating the polymer's association with water. Notably, the intensity of this peak is particularly high in the case of Ca2+ due to its strong affinity for water and its network. Furthermore, the observed peak at 96 in the mass spectrum provides confirmation of the presence of the [SO4]+ fragment. The presence of [120]+ (SO3Ca)+ and the inference of (133 − 77) = [56]+ provide evidence supporting the existence of the [OCa2+] fragment. This comprehensive analysis verifies the identification of carrageenan as the ion carrying the Ca2+ ion. Furthermore, the characteristic base peak observed at [207]+ provides additional confirmation of the carrageenan skeleton.
Optimization of the solvent:
The choice of solvent can influence how carrageenan and interacts with other components. Common solvents include water and organic solvent were screened to analyse the gelling properties of carrageenan, The carrageenan with divalent cations like Ca²⁺ and its status in different solvent were noted as depicted in the below Table 1.
Table 1
Solvent Screening and Solubility Profile of carrageenan.
S. No.
|
Solvents
|
Observation
|
1
|
Ethyl Acetate*
|
Not Forming Gel and Insoluble
|
2
|
Ethanol
|
Not Forming Gel and Insoluble
|
3
|
Dichloromethane (DCM)
|
Not Forming Gel and Insoluble
|
4
|
N, N, dimethyl Formamide (DMF) *
|
Not Forming Gel and partially soluble
|
5
|
Acetone
|
Not Forming Gel & Insoluble
|
6
|
Toluene*
|
Not Forming Gel and Insoluble
|
7
|
Acetone
|
Not Forming Gel and Insoluble
|
8
|
Dimethyl sulfoxide (DMSO)*
|
Not Forming Gel and Insoluble
|
9
|
Methanol
|
Not Forming Gel and Insoluble
|
10
|
Acetonitrile*
|
Not Forming Gel and Insoluble
|
11
|
Water
|
Not Forming Gel and Insoluble
|
12
|
Water (Hot)
|
Gel formation
|
* Temperature was also enhanced till boiling point and similar observation was observed.
The solubility of the Ca+ 2 Carrageenan was tested using polar protic (Acetone, Alcohols), aprotic (DMF), high boiling polar solvent (DMSO), and non-polar (toluene) solvents; however, none of the solvents were successful in producing the carrageenan solution phase, Table 1 (Entry 1–10), even after raising the temperature to the boiling point of the corresponding solvent. It was anticipated that the polymer would remain in solution phase following dissociation, which was only made possible in water due to the solvent's high dissociation capacity. After screening with organic solvents, water (Table 1 entry 11) was utilized as an aqueous solvent. Surprisingly, it wasn't solvable, and the outcomes from entries 1 through 11 were comparable as before.
The temperature of the carrageenan solution in water was gradually raised until it took the form of a gel; however, in the case of K+, the gel form was with water at a normal temperature and the solution phase was with a high temperature; the opposite result with Ca2+ provided a reason to further optimize the temperature effect to see how the behaviour changed as the water temperature changed.
Table :2 Effect of temperature in the water and carrageenan system:
Ice-Water
|
Normal Water
|
Hot water
48-80oC
|
High Temperature 80oC
|
Not Forming Gel, Insoluble
|
Not Forming Gel, Insoluble
|
Gel formation
|
Viscous solution, soluble
|
The temperature-based analysis shows the pattern of Gel-sol property after the heating, however in case of K+ the sol-Gel conversion was taking place with respect to the heating. The further investigation of the ion- effect was caried out, by keeping in the mind the Ca2+is smaller (2.4 Å) than the potassium (2.7Å) and the computational based study regarding the distance between sulphate ion and K+ and Ca2+ as depicted in Fig. 6.
The distance between Ca2+ and carrageenan was less than the K+, it was also clue regarding the more attraction or more bond dissociation energy with the Ca2+ion. It was anticipated that the cold/normal water does not converts in to the ionic form, however the high temperature when matches with the dissociation energy gets in to the dissociation for and forms gel with stable 1C4 conformers for the formation of gel. It was also interesting to see that heating more than 80oCwas converting Gel to solution, because of its conformational change from 4C1, A higher energy state for solution as it is related with the reported study regarding the role of conformational changes and its effect on phase, as depicted in the below Fig. 6. Moreover, the network with H2O which was observe in the IR and Mass also suggest the close linkage of the network in undissociated form, which does not get ionized in the normal temperature so the higher temperature is require to form the gel in with stable 1C4 conformer as depicted in Fig. 7.
However, in the case of K+ the bond attraction was less so it dissociated in normal water and forms gel, and in case of the hot water it forms solution because of the conformational change to 4C1 as solution phase. Figure 8
The dissociation theory for Ca2+ was further analyse through the ionic content in the solution with respect to the temperature. The carrageenan and water solution were reacted with the NaOH solution in each temperature and the pattern of the reactivity was found different as per given Table 3
Table 3
Reaction of free calcium ion with NaOH in different temperature.
Ice-Water
|
Normal Water
|
Hot water
48-80oC
|
High Temperature 80 oC
|
After the addition of NaOH, the solution turns viscous.
|
After the addition of NaOH, the solution turns more viscous
|
After the addition of NaOH solution, it does not give any change
|
After the addition of NaOH solution, it does not give any change
|
Very light Precipitate forms after 15–20 mints with less yield
|
Light precipitate forms in 15 minutes
|
Precipitate in few minutes with high yield
|
Precipitate in few minutes with high yield
|
The reactivity pattern with NaOH clearly represent the availability of free calcium ion present in the solution, it also confirms the dissociation of the Ca2+ is also increasing with high temperature and justifying our plausible mechanism regarding the undissociated form in cold water of Ca-linked carrageenan. The ionic content in hot water was also confirm by the conductivity as the io gets dissociated the conductivity increase and the similar result obtained in the test, and the gelling property correlation with ionized and unionized form was confirm through the experiment. Conductivity based on the principle by measuring the electric current flows in electrons present in the solution means if the solution is higher in ion concentration shows reading towards higher, which is observed in our result, as ca ion are in dissociated form in hot water so showing conductivity more than cold and normal water. However. The conductivity of the K+ carrageenan remains same in all the condition, so the dissociation was same in the all cases, while with the Ca2+it increases with the temperature.
Table 4
conductivity of the water carrageenan solution in different temperatures.
Ca CG with water at different temperature
|
Readings
|
Hot water (50-1000C)
|
0.59–0.64
|
Water (RT)
|
0.01
|
Cold water (8 0C) Normal
|
0.01
|
The investigation into the Ca2+ linked carrageenan and its temperature-dependent role is supported by a number of chemical and experimental evidence, and it is suggested that because the Ca2+ linked carrageenan has a higher dissociation energy than the K+ linked carrageenan, it forms gel in hot water and is insoluble in cold water, supporting the role of cations and conformational changes.
Viscosity:
The viscosity of the K + linked gel was found as 649 centipoise and the gel with Ca2+ has 728, (Table 5) which also justifies the higher cross linking with helical-to-helical linkage to form more stiff and stable gel. The intermolecular interactions (Fig. 9) were also observed by IR through the OH deformation peak, so the more interactions get more stiff gel.
Table 5
Viscosity results of nano and simple gel
Type of Gel
|
Viscosity
|
K+Carrageenan gel
|
649
|
Ca+Carrageenan gel
|
728
|