Physicochemical study of keratin containing impurities from human nails

The purpose of this research to study of physicochemical properties of keratin from human nails which still contains impurities. The method in this study, keratin from human nail waste was dissolved using NaOH solvent at room temperature, then neutralized to pH 7 using glacial acetic acid. On this report have studied the analysis of functional groups of keratin with impurities, viscosity, melting point, solubility and diffractogram pattern using XRD. The FTIR spectrum shows the transmittance in the wave number 1750-1200 cm−1 which is the characterization region of keratin protein. The intrinsic viscosity of nail keratin was obtained at 0.105 g/dL or 10 g/ml. The melting point of nail keratin was obtained at 210 °C. Keratin solubility test using HCl and NaOH 0.1 M and 1 M respectively, showed that keratin was soluble in strong base NaOH with high concentration. The results of XRD analysis, in a diffractogram pattern without a dominant peak and tend to widen, keratin from human nails is included in the amorphous structure type.


Introduction
Nails are composed of protein material, namely keratin.Keratin is a scleroprotein (fibrous protein) that contains large amounts of sulphur to form the tissues of animal horns, hooves, feathers, and the like.Polypeptide chains are the basic macromolecules that make up keratin.This chain consists of two, namely the curved helical conformation (α-keratin) and the adjacent lipid conformation (β-keratin).In mammals, keratin is the main constituent of hair, nails, spines, horns and the epidermal layer of the skin.(α-keratin) or hard keratin, embryologically derived from epithelial cells which are the main constituents of keratinized mammalian tissues such as nails, horns, spines and the like, while (β-keratin) or soft keratin is the main component of the epidermis (skin surface).Human nails consist of about 80% hard keratin and 20% soft keratin [1].The polypeptide chains in keratin are formed by the condensation of several amino acids.Although they are all derived from ectodermal cells, there are fundamental physical and chemical differences in the very different shapes of nails, hair, and stratum corneum.Hair and nails are distinguished along the same lines although their morphological characteristics are different.Nails contain 10-20% more basic soft keratin, in addition to the sulphur-rich hard keratin present in both tissues [2].Significant proportions of glutamic acid, half cystine, arginine, aspartic acid, serine and leucine are present in the human nail plate.The content of glycine and half cystine is similar in human hair and nails, but not in the stratum corneum.An extraordinary degree of physical and chemical stability is afforded to the keratin filaments by the extensively cross-linked disulphide structure corresponding to the high proportion of cystine [3].Many studies related to keratin have been reported, but in terms of physicochemical data, there are still few reports [4][5][6].Based on this percentage, this research will be carried out by taking random samples of human nails to make nail powder containing keratin in order to determine the physicochemical properties of the isolation results with characterizations that include FTIR, intrinsic viscosity, melting point, solubility and XRD.

Experimental 2.1 Sample Preparation
The materials used were human nails collected from friends, relatives, close friends, as well as beauty salons, NaOH, HCl, acetone, glacial acetic acid, detergent, and distilled water.The sample preparation carried out in this study was the waste of human nail clippings washed with water and detergent repeatedly to remove adhering dirt.After the washing process, the waste of human nail pieces is dried.After drying, the nail sample was immersed again in acetone solution for 30 minutes to remove the oil contained on the nails.Next, the nails are cut into small pieces using scissors.Furthermore, the prepared nails were soaked as much as 30 grams into 150 mL of 1 M NaOH solution until completely dissolved.Then added glacial acetic acid which is used to neutralize the solution to (pH 7) of approximately 6 ml.Then the solution was heated at 70 °C on a hotplate and stirred until a gel was formed.

Sample Characterization
Characterization of keratin powder from human nails using FTIR for the analysis of keratin functional groups using a wave number range of 4000 to 500 cm -1 .Intrinsic viscosity was determined using the Ostwald viscometer.Determination of the relative molecular mass of the polymer can be used by comparing the viscosity of the solvent with the viscosity of the polymer solution.To determine the value of intrinsic viscosity, then made a relationship between viscosity reduction vs. C (concentration).The point that intersects the Y axis is the intrinsic viscosity value.In testing the melting point of the nail keratin sample, the sample is inserted into a capillary tube approximately 2 cm long, then put into the melting point.Keratin powder from human fingernails and their temperature were observed by reading the thermometer on the melting block when the substance in the capillaries started to melt [7].In the solubility test of samples from human nail powder, the solvents used were HCl and NaOH with varying concentrations of 0.1 M and 1 M. Keratin crystallinity test was tested using XRD which resulted in a curve showing the relationship between intensity and 2θ.XRD curve analysis produces a diffractogram pattern on nail keratin.

Results and Discussion
The manufacture of nail powder containing keratin has carried out by dissolve the nail powder as much as 30 grams into 150 mL of 1 M NaOH solution.The nail solution is stirred until completely dissolved.Glacial acetic acid added to a solution of 6 mL aims to neutralize the solution using pH indicator paper so that pH 7 is obtained in a neutral state.From figure 1, the addition of acetic acid also causes a phase change to become a gel so that the solution becomes more viscous and the color changes to greenish brown.This happens because of an acid-base reaction between a strong base NaOH and a weak acid from glacial acetic acid which produces sodium acetate and H2O.Furthermore, the solution was heated at a temperature of 70°C on a hotplate and stirred until it became a gel and was easily dried and then in a blender to produce nail powder (Figure 1, b).The final weight obtained after making the powder is 25.91 grams.Keratin powder is not left in the open air considering its hygroscopic nature, so it still contains water vapor and produces peaks of hydroxyl functional groups (-OH) [8].Characterization of keratin powder from human nails using FTIR aims to identify the functional groups of the compounds that make up the material and their purity, the spectrum of the analysis results has showed in figure 2. The range of wave numbers used for the analysis of human nail keratin functional groups is 500 cm -1 up to 4000 cm -1 .From the spectrum, region in the wave number 1750-1200 cm -1 is the characterization region of the protein keratin, in which keratin is a long chain of amino acids linked together by amide bonds I, II, III.The region around the wave number 1642 cm -1 is amide I (C=O stretch), the region around the wave number 1551 cm -1 is amide II (stretched CN and -NH bending) and the region around wavenumber 1247 cm -1 shows as CH stretching and NH bending.Other regions, namely at wavenumber 1395 cm -1 is the CH bending region containing fatty acids and lipids, the transmittance peak seen around wavenumber 1124 cm -1 is the vibration of the stretch COC bond, CS stretching vibration (Sulphide) at absorption 825 cm -1 [8][9].The table comparison of functional groups between keratin left hand middle nail sample and keratin human nail powder showed in Table 1.

Table 1. Comparison of functional groups between keratin left hand middle nail sample and keratin human nail powder
Intrinsic viscosity was determined using the Ostwald viscometer.Ostwald viscometer is a tool used to measure the viscosity or viscosity of a polymer solution.Determination of the relative molecular mass of the polymer can be used by comparing the viscosity of the solvent with the viscosity of the polymer solution.825 839 Determination of the intrinsic viscometer has been done by determining the flow time of keratin from nail powder using mass variations, namely 2 g/dL, 4 dL/g, 6 dL/g, 8 dL/g and 10 dL/g.The flow time of each mass was carried out with 3 repetitions where sequentially the average flow time was obtained, namely 219 seconds, 260 seconds, 302 seconds, 346 seconds and 393 seconds.After obtaining the polymer flow time with the solvent, then a graph of the relationship between concentration and reduced viscosity (ƞsp/C) was made (Figure 3).Based on the graph, the intercept value is obtained, namely 0.105 dL/g.This value is the intrinsic viscosity [ƞ].The Intrinsic viscosity is directly proportional to the flow time, the greater the viscosity, the more difficult it is for a solution to flow so it takes more time to flow.The melting point is the temperature at which a compound begins to change phase from a solid to a liquid, until complete melting occurs.In another sense, the melting point can also be interpreted as the temperature at which a solid will be change turns into a liquid at a pressure of one atmosphere [10].In testing the melting point of the keratin sample from nail powder, the sample is inserted into a capillary tube approximately 2 cm long, then put into the melting point.After a few minutes, the human nail sample showed a change of state to liquid at a temperature of 210°C as shown in Figure 4.This corresponds to the nature of keratin which has a melting point above 200°C.The stronger the bond of keratin, the more energy is used to break the bond.This leads to a higher melting point [11].In the solubility test of keratin samples from human nail powder, the solvents used were HCl and NaOH with varying concentrations of 0.1 M and 1 M. Samples that had been weighed as much as 0.5 grams were dissolved in 7 mL of solvent and then stirred for 2 minutes.For the solubility test using 1 M HCl as a solvent, it showed that the sample was clotted/not completely dissolved and the resulting colour was bright yellow, while for 0.1 M HCl it showed that the keratin still contained dispersion or sample granules and the resulting colour was brownish yellow.
For the solubility test using 1 M NaOH solvent, it shows that the keratin dissolves completely without showing any lumps and the resulting coloured is brownish yellow, while in 0.1 NaOH, the keratin dissolves completely and is dark brown.The solubility test of keratin samples from human nail powder shown in Figure 5.The isolated keratin powder was characterized using XRD.XRD analysis aims to determine the presence, purity, crystallinity and size of keratin in nails.Crystallinity can affect performance and Xray diffraction determination of crystallinity.The results of the XRD test are in the form of a curve that shows the relationship between intensity and 2θ.XRD curve analysis yielding a diffractogram pattern on nail keratin has showed in figure 6.

Figure 6. Human nail keratin XRD diffractogram
The results of the diffractogram show the characteristic peaks of human nail keratin.The crystal structure characterization of keratin was observed at an angle of 2θ = 0°-100°.The highest peak at the value of 2θ is equal to 20.37°.In this pattern, it does not show any dominant peaks in each structural plane, in other words, based on the resulting structure, keratin from human nails belongs to the amorphous structure type.The amorphous phase causes more empty space which allows more ion movement to occur.The resulting structure of nail powder containing amorphous keratin has an irregular and not dense structure, while the crystalline structure has regularity [12].

Conclusion
Based on the results of this study, several conclusions can be drawn as follows: the manufacture of human nail keratin has been carried out using the hydrolysis method using NaOH solvent and acetic acid at a temperature of 70°C, resulting in a hygroscopic keratin powder that produces a hydroxy functional group (-OH).Characterization of keratin from human nail keratin using FTIR showed the presence of functional groups, including the absorption peak at wave number 1642 cm -1 was amide I (C=O stretch).Absorption peak at wave number 1551 cm -1 is amide II (stretched CN and -NH buckling) and the absorption peak at wave number 1247 cm -1 (stretched CH and bent NH) is amide III.The intrinsic viscosity of keratin obtained from the intercept is 0.105 g/dL.The melting point of nail keratin is 210 °C.Keratin solubility test showed that nail keratin was soluble in strong alkaline solvent NaOH.Characterization using XRD resulted in amorphous form.

Figure 3 .
Figure 3.The relationship between concentration and reduced viscosity.

Figure 4 .
Figure 4. Process of determining the melting point of insulation from human hair

Figure 5 .
The solubility of human nail keratin in (a) HCl and (b) NaOH.