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This experiment was designed to investigate and compare the extent of damage to the hair shaft structure caused by henna as the selected natural hair dye and chemical hair dye. The qualitative analysis of hair using a hair hygrometer shows the variation in hair strand length in a fixed moist condition differ between the naturally and chemically dyed hair. The mean length of extension represents the degree of damage of the hair as the hair dyes interfere with the hair shaft structure, causing more water to be absorbed into the hair shaft. The statistical t-test with significance level of 0.05 showed that the mean extension length of the chemically dyed hair is significantly larger than that of the naturally dyed hair. The results support the experimental hypothesis that chemically dyed hair is more damaged than naturally dyed hair.
Research and Rationale
Hair colouring is a chemical process involving a series of complex reactions between chemicals in the hair-colouring product with chemicals and biochemical in hair to change the hair colour. The chemicals prepare the hair to accept the hair dye, alter the hair shaft biology to maximize colour change and set the dye to make the colour change permanent. 
The hair shaft is composed of two major components - the cortex (largely keratins) and the cuticle (made up of thin scales of dense keratin and a lipid coating).  To change hair colour, the protective oils on the hair shaft have to be partially removed by a bleaching agent, commonly hydrogen peroxide that oxidizes the melanin pigment in hair, making it colourless.  The most common chemical hair dyes used are para dyes, containing paraphenylenediamine (PPD) solutions which combine with hydrogen peroxide to create insoluble molecules which are contained within the cortex and are unable to pass through the cuticle layers, leaving colour to the hair.  ,  Due to the bleaching agent and ammonia present in dyes, changes in the structural keratins and protective oils make the hair shaft drier and more brittle, weakening the whole hair structure.
Natural substitutes have been explored to minimize the risk of side effects of chemical hair dyes. In fact, many natural agents like plants and minerals have been used for hair colouring for thousands of years. These agents contain natural bleaching agents that change hair colour by coating the hair shaft with colour.  For example, henna (Lawsonia inermis) is a small tree growing to six meters high. Its colouring properties are due to the presence of lawsone, an organic compound that has an affinity for bonding with protein.  This compound is highly concentrated in the leaf petioles and gives a red-orange colour. Natural hair dyes like henna are non-toxic but some believe that they are not necessarily safer or gentler than modern formulations as consistent results are hard to be obtained and allergic reactions may arise in different individuals.
Therefore, this experiment was aimed to compare the extent of damage to the hair shaft caused by natural and chemical hair dye. In this experiment, the extents of damage caused by respective dyes are compared in terms of their response to moisture in the air. Hair expands as air humidity increases and vice versa. Due to the damage by hair dyes, the ability of the hair strand to absorb water is affected.
The results from this study can be used to show that chemical hair dyes alter the hair shaft structure more than natural hair dyes. This proves that natural hair dyes are able to provide a non-toxic choice for hair dyeing that produces good results. Hence, this also eliminates the risks of harmful chemicals present in the chemical hair dyes whereby some carcinogenic chemicals believed to be present has become a great concern.
There is a significant difference between the damage to hair structure due to chemical hair dye and natural henna dye. Chemical hair dye causes more damage to the hair shaft structure compared to the henna dye.
There is no significant difference between the damage to hair structure by chemical and natural henna dye.
Manipulated : Types of hair dye
Responding : Mean length of hair extension
Fixed : Condition of hair strands before dye-ing, length of hair on hygrometer, humidity in bell jar, temperature of surroundings
Apparatus and Materials
Boar bristle brush, plastic gloves, two 250ml beakers, hair colouring brush, glass rod
Pestle and mortar, sift, 10ml measuring cylinder
50g henna leaves, vinegar
Chemical Hair Dye
50ml permanent cream hair colour (Ingredients: aquq, stearyl alcohol, ammonium hydroxide, isostearic acid, oleth-10, propylene glycol, ammonium laurl 3ulphite, dehydrol 2409, merouat plus 3330, sodium 3ulphite, sodium erythorbate, tetrasodium edentate dehydrate, perfume, p-phenylenediaminesulfate), 6% cream peroxide
Wood piece (7.5cm X 20cm), plastic paper, 2 small nails, dime, tape, hammer, scissors, bell jar, two 100ml beakers, metre rule, vernier caliper
Hair strands (undyed, henna dyed and chemically dyed), distilled water
Table 1: List of apparatus and materials used in the experiment
Determining observation method & modification
Trial experiments were conducted to obtain the best observation method to compare the extent of damage between natural and chemical hair dye.
Due to ammonia present in hair dye, the hair shaft swells, forcing the cuticle scales to separate slightly. The corrosive property of ammonia wears away the hair shaft. Under the microscope, hair treated with ammonia will resemble Swiss cheese, according to the Organic Colour Systems website.  Hence, the number of breaks on the hair strand could be counted under the microscope to compare the damage caused by natural and chemical hair dyes. The hair strands were placed onto a microscopic slide. About two drops of distilled water were dropped onto the slide. The hair strands were viewed under microscopic power 40X. An ocular and a stage micrometer were used to measure the length of strand. Two strands were observed for natural and chemical hair dye respectively. (Appendix Figure 2)
Due to the loss of sebum on the outer layer, dyed hair loses ability to repel water. Instead, water is absorbed, causing the hair to be more fragile. Thus, the degree of damage by hair dyes can be compared by measuring the extension of hair strands under fixed moist conditions using a metre rule. Furthermore, a control group using un-dyed hair strands was included to indicate that the damage were significantly shown through the extension of hair length on the hygrometers.
Calibration Value (Âµm)
Stage micrometer : Number of mm
Eyepiece graticule : Number of intervals
40 eyepiece units
Results for both methods are shown in the tables below.
Number of eyepiece units
Length of strand (mm) = number of eyepiece units X calibration value
Number of breaks
Number of breaks on strand for every 10mm
Table 2: Number of breaks on hair strand with respect to hair dyes
Mean Length of Hair Extension (mm)
Table 3: Mean extension in length of hair with respect to hair dyes
Based on the results above, the hair hygrometer method was selected as it showed significant difference of damage on hair strand between henna dye and the chemical dye. There were some breaks of the hair strand that were ambiguous, therefore whether to take into account a break or not depends on the observer. The reliability of the microscopic method is thus less compared to the hair hygrometer method.
The metre rule used to measure the length of hair extension in the hygrometer method has only accuracy up to 0.1cm. Hence, to improve accuracy of the results as the readings are small, a vernier calliper measuring up to 0.02mm was used.
Appendix Figures 2, 3, 4
Harvesting hair for dyeing
Un-dyed hair strands were collected from ten female samples.
The hair strands were shampooed and rinsed with water to remove any impurities.
The hair strands were divided into 2 groups - naturally and chemically dyed group respectively.
Preparing henna dye
Using pestle and mortar, dried henna leaves were grind evenly into powder and sifted.
The sifted powder was transferred to a 250ml beaker and 10ml of vinegar was added to hasten the dye release process.
The mixture was stirred with a glass rod until the henna powder is completely dissolved and a thick paste was formed. Vinegar was further added until the desired creamy consistency was reached. The paste was left aside for 24 hours to allow the henna dye to be completely released.
Dyeing hair strands using henna
Hair strands secured at one end with tape were placed mixed with the henna paste using glass rod.
A brush was used to brush the hair strands to ensure even distribution of dye on the strands.
The beaker was left aside for 4 hours then rinsed with water thoroughly and left to dry.
Dyeing hair strands using chemical hair dye
The 50ml permanent cream hair colour and 6% cream peroxide were mixed thoroughly using a glass rod in the 250ml beaker.
Repeat steps 7 to 9 using the chemical dye but leave only for 45 minutes.
Preparing the set up of the hygrometer
The plastic paper was cut into a triangular shape to make the pointer of the hygrometer. The dime was taped onto the plastic near the point. One nail was poked through the plastic pointer near the base of the triangle. The other nail was nailed on the upper part of the wood piece.
The pointer was nailed 12.5cm away from the first nail onto the wood piece. The pointer should move freely and loosely about the nail.
Three natural dyed hair strands were taken. One end was taped to the nail on the upper part; the other onto the pointer. The strand ends that were too long were trimmed. It was made sure that the hair strands hung perfectly vertical from the nail to the pointer. The hair strands were pulled straight and tight so that the pointer points parallel to the ground (perpendicular to the hair).
Steps 12 to 14 were repeated so that two hygrometers were made using henna-dyed and chemically dyed strands respectively.
Recording Observations using Hygrometer
The initial point on the wood piece at which the pointer points was marked. The two hygrometers were placed inside a bell jar each with two 100ml beakers of distilled water for 3 hours respectively.
After 3 hours, the new point at which the pointer points was marked and the length of the pointer from its original point was measured.
Steps 16 to 17 were repeated using new sets of hair strands to obtain ten sets of results.
As chemicals were involved, protective gloves were worn during the hair dyeing process. Henna dye may cause allergic reactions on skin of particular individuals while the bleaching agent, peroxide cream present in the chemical hair dye is corrosive. Hence, protective wear were used to minimize contact with skin and eyes.
All the glassware used including bell jar, measuring cylinders, glass rods and beakers must be handled with extra care as they are easily breakable and may cause injury. Besides that, the hair strands might break easily so extra care is taken when the hair strands were attached to the hygrometer.
Length of extension of hair(mm)
Table 4: Lengths of extension of hair with respect to hair dyes.
Figure 5: Bar Chart of mean length of hair extension with respect to hair dyes
The t-test was chosen as the statistical test for the experiment to show the significance of difference between the means of two independent samples (natural and chemical hair dyes.)
xÌ„ = âˆ‘x/n
â”‚ xÌ„1 - xÌ„2â”‚
â”‚ 4.803 - 9.339 â”‚
âˆš (s12/n1) + (s22/n2)
âˆš (0.175341/10) + (1.137019/10)
xÌ„1 = mean of sample 1 (henna dye)
xÌ„2 = mean of sample 2 (chemical dye)
n1 = number of subjects in sample 1
n2 = number of subjects in sample 2
s12 = variance of sample 1
s22 = variance of sample 2
degrees of freedom, df = n1 + n2 - 2 = 10 + 10 - 2 = 18
significance level, Î± < 0.05
tabulated t-value = 2.101
calculated t-value = 12.52
Therefore, ttable < tcalculated
Table 5: Calculations for t-test
The mean length of extension of hair dyed with chemical dye is significantly longer than that dyed with henna. The calculated t-value (12.52) shows that it is significant whereby it exceeds the tabulated t-value (2.101). Therefore, the experimental hypothesis is accepted and the null hypothesis rejected.
The mean extension of the chemically dyed hair is 4.54mm longer than that of the henna-dyed hair. This gives a percentage difference of 48.6% between both dyes. Error bars are displayed on the bar chart to relay 95% confidence level in the data. The ranges in both dyes do not overlap; meaning that the two mean values are significantly different. Hence, it suggests that the experimental data is sufficient to prove the experimental hypothesis.
The control uses un-dyed hair strand. It showed change in length, indicating that hair is hygroscopic. This is achieved by capillary action whereby water vapour diffuses through the minute spaces of the hair despite the close fitting scales of cuticle and sebum.  This is the basic working principle of the hair hygrometer. A relative small change in the control group proves that hair dyes affected the hair structure and permeability to water.
The hair dye alters the protective oils on the cuticle layer,4 so water vapour diffuses easily into the hair, causing it to swell.  The extension in hair length was shown by the movement of the hygrometer pointer. As the hair strand extended, the pointer moved downwards, pulled by the weight of the dime. The less damaged the hair structure is, less water is able to diffuse into it in a fixed time. Thus, the extension in hair length is a measure of the hair dye's damaging property.
The percentage increase in length of the chemically dyed hair is 7.5% while that of henna dyed hair is 3.8%. This corresponds to the information from source  whereby it states that a hair in good condition absorbs more than 30% of its own weight of water; for damaged hair, this percentage reaches 45%, thus length increases up to 2%.
Loose hair was taken from several female subjects so the conditions of the hair strands before dyeing need to be constant: hair strands collected were completely natural and un-dyed; short hairs less than 12.5cm were discarded; care taken to ensure that the strands did not break when being extracted from the hair brush. Furthermore, hair strands were cleaned before dyeing so that any oil or impurities on the strands do not affect the results. Three strands were used on the hygrometer as a single strand is easily broken.
However, the length of dye time for both dyes could not be fixed the same. This is because for optimum dyeing, henna dye requires a longer time as it does not contain chemicals which are able to react fast to penetrate the cuticle.
There may be errors due to the limitations of the hair hygrometer. Hair expands or shrinks not only in response to humidity but also temperature. The expansion or shrinking corresponding to a temperature change of 1oC is about 1/15 of that corresponding to a temperature change of 1% in usual air temperatures.  Hence, surrounding temperature has to be kept constant at all times so that this factor does not affect the validity of the results. The hair hygrometer is a mechanical instrument which contribute to errors like 'setting' of the hair strand if remained at a constant humidity for prolonged period of time and slow response times.  Therefore, a suitable period of time needs to be determined to minimize both these errors.
The experiment could be modified by measuring the diameter of the hair strand under microscope to obtain results. The hygrophobic property of hair can not only be measured in terms of length but also diameter. According to source , damaged hair can increase by 15% to 20% in diameter. The results obtained would be more accurate as hair strands are measured singly. Furthermore, the tensile strength of hair can be investigated as hair dyes affect the strength of the hair shaft. An extensiometer can be used to progressively stretch the hair at the rate of 1cm/minute, allowing precise measurement and comparison of the breaking point of dyed hair.
Chemical hair dyes are significantly more damaging to the hair shaft compared to natural hair dyes. This was determined by the mean extension of the dyed hair on the hygrometer. There is a statistically significant difference between the mean extension length of hair and the extent of damage of natural and chemical hair dyes. Experimental hypothesis is accepted and null hypothesis is rejected.
Source 12 is an online journal obtained from the online database from my campus library. ScienceDirect is a leading full-text scientific database offering more than 2500 peer-reviewed journals and more than 11000 books. There are currently more than 9.5 million articles/chapters. Hence, this is a highly reliable source.
I have also obtained information about hair dyeing from a range of sources. Source 1, Hair Foundation is an organization that provides credible and the most recent scientific information on hair health to the public. It is considered a trusted source as the other cross-referred websites (sources 3 and 5) have similar and relevant facts as source 1.
The online journal (source 2) is another dependable source as the writer, Desmond J. Tobin, a medical biosciences PhD holder from the University of London, UK (St. Thomas's Hospital Medical School), is experienced in this topic and cited a lot of trustable references. This article is published by the Royal Society of Chemistry, one of the largest publishers of chemical science information in the world. The quality content of the books, databases, journals and magazines are thus very reliable.