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J. Cosmet. Sci., 67, 1–11 (January/February 2016)
1
Investigation of hair dye deposition, hair color loss,
and hair damage during multiple oxidative dyeing and
shampooing cycles
GUOJIN ZHANG, ROGER L. MCMULLEN, and
LIDIA KULCSAR, Ashland, Inc., Bridgewater, NJ 07004.
Accepted for publication October 5, 2015.
Synopsis
Color fastness is a major concern for consumers and manufacturers of oxidative hair dye products. Hair dye
loss results from multiple wash cycles in which the hair dye is dissolved by water and leaches from the hair
shaft. In this study, we carried out a series of measurements to help us better understand the kinetics of the
leaching process and pathways associated with its escape from the fi ber. Hair dye leaching kinetics was
measured by suspending hair in a dissolution apparatus and monitoring the dye concentration in solution
(leached dye) with an ultraviolet–visible spectrophotometer. The physical state of dye deposited in hair fi bers
was evaluated by a refl ectance light microscopy technique, based on image stacking, allowing enhanced depth
of fi eld imaging. The dye distribution within the fi ber was monitored by infrared spectroscopic imaging of hair
fi ber cross sections. Damage to the ultrafi ne structure of the hair cuticle (surface, endocuticle, and cell membrane
complex) and cortex (cell membrane complex) was determined in hair cross sections and on the hair fi ber surface
with atomic force microscopy. Using differential scanning calorimetry, we investigated how consecutive coloring
and leaching processes affect the internal proteins of hair. Further, to probe the surface properties of hair we
utilized contact angle measurements. This study was conducted on both pigmented and nonpigmented hair to
gain insight into the infl uence of melanin on the hair dye deposition and leaching processes. Both types of hair
were colored utilizing a commercial oxidative hair dye product based on pyrazole chemistry.
INTRODUCTION
Hair dyeing is a common cosmetic practice in the beauty industry and is carried out in the
salon by professional cosmetologists as well as at home by the consumer. It is an especially
common procedure for individuals with gray hair, but is also practiced by members of the
younger population. Usually, hair is periodically dyed (every 4–6 weeks) due to new hair
growth. The most suitable kinds of coloration come from the use of oxidative (or perma-
nent) dyes, which offer a great variety of shades and increased wash fastness. In general,
oxidative dyes consist of primary intermediates, color couplers, and oxidizing agents (1,2).
During the coloring process, the primary intermediates are activated by hydrogen peroxide
and then react with color couplers inside the hair shaft. The newly formed molecules inside
Address all correspondence to Roger McMullen at rmcmullen@ashland.com.
JOURNAL OF COSMETIC SCIENCE2
the hair are colored and large enough so that they do not easily leach out from the hair
structure. They also have affi nity to hair, presumably due to van der Waals or other nonco-
valent interactions between the dye and the internal structural components of the hair, al-
lowing them to remain in the hair structure even during shampooing or rinse out (3).
Hair dye deposition and water fastness is infl uenced by the permeability of primary inter-
mediates into the hair and the health condition of the ultrafi ne structural components of
hair. Typically, oxidative hair dyeing is carried out at high pH (~10), utilizing ammonia
or ethanolamine, causing cuticle swelling and facilitating entry of the dye intermediates
into the fi ber structure as well as to decompose hydrogen peroxide so that the dyes can be
activated (4). This high pH damages many of the lipids on the surface of the hair, making
it a more hydrophilic substrate with greater capacity to absorb ingredients (5). In addi-
tion, bleaching, dyeing, and other harsh chemical treatments damage the ultrafi ne struc-
ture of hair leading to an overall more porous, or open structure, allowing dye molecules
to easily diffuse into and out of the hair (6–8). The ease with which a molecule diffuses
into or out of the fi ber is dependent on a number of factors including its molecular size
(9). In fact, kinetic studies of dye removal by Wong et al. demonstrated that smaller dyes
rinse out much more readily than larger dyes (10).
In addition to surface damage, oxidizing agents in permanent hair dye systems also dis-
solve melanin and oxidize hair keratin substrate (8). Therefore, both the hair surface bar-
rier of the cuticle and the ultrafi ne structure of the cortex are greatly changed. To date,
there has not been a comprehensive study on the mechanism of hair dye deposition and
leaching pathways published in the literature due to the complexity of hair damage caused
by the dyeing process. In this study, we investigated hair damage by consecutive dyeing–
shampooing equivalent cycles and elucidated dye deposition and leaching pathways. This
information will be helpful for scientists to develop improved technologies that minimize
the amount of damage in the coloring process, prevent color fading from washout, and
even create specialized products that restore health and brilliance to colored hair.
MATERIALS AND METHODS
A number of experimental procedures and instruments were used to gain a better under-
standing of the dye leaching process and the damage associated with the bleaching and
dyeing of pigmented and nonpigmented hair. As already mentioned above, instrumental
techniques were employed to investigate the morphological characteristics of the ultrafi ne
structure of hair, and consisted of atomic force microscopy (AFM), differential scanning
calorimetry (DSC), and dynamic contact angle analysis. In addition, we monitored the
deposition of dye within the fi ber structure using refl ected light microscopy and Fourier
transform infrared spectroscopy (FT-IR) spectroscopic imaging. Further, studies of the
kinetics of dye leaching were carried out by exposing dyed hair to a number of rinse cycles
with water while monitoring the aqueous dye concentration.
HAIR TRESS PREPARATION
The experiments were conducted on both European white and dark brown hair purchased
from International Hair Importers (Glendale, NY). Hair tresses were prepared by gluing
~2 g of fi bers together at the hair root to a Plexiglas tab with Duco cement. The resulting
dimensions of the hair tresses were 6.0 inches in length by 1.25 inches in width. Hair
HAIR DAMAGE DURING MULTIPLE OXIDATIVE DYEING AND SHAMPOOING 3
tresses were precleaned with a 3% ammonium lauryl sulfate solution, rinsed thoroughly,
and dried prior to use in the experiments. Hair was then subjected to a bleaching regimen
for 30 min with Clairol BW2 (Procter & Gamble, Cincinnati, OH) beaching powder and
20-volume hydrogen peroxide (Clairoxide 20; Procter & Gamble).
MULTIPLE HAIR DYEING–LEACHING CYCLES
In this study, hair was subjected to a regimen that consisted of a dyeing step followed by
a dye leaching step, which was carried out by immersing freshly dyed hair fi bers in water
for 30 min at 40°C (called one cycle). Five dyeing–leaching cycles were conducted on
both white and dark brown hair. During the dyeing steps, 12 hair tresses were dyed with
Textures and Tones 4R (Red Hot Red) hair dye (Procter & Gamble, Cincinnati, OH) for
40 min. Hair tresses were then rinsed for 2 min under hot water (~38°C) and excess water
was removed by forming a squeegee with the index and middle fi ngers and running them
along the length of the tress. Hair tresses were then dried with a hair blow dryer (tem-
perature set to medium). During the dye leaching process, each freshly dyed hair tress was
suspended in a vessel containing 500 ml of water at 40°C with continuous stirring at
50 rpm. The total dye leaching time is 30 min. Leached dye was measured with an auto-
mated dissolution system, consisting of a VK7000 dissolution testing apparatus (Varian
Inc., Cary, NC) and an ultraviolet–visible spectrophotometer equipped with seven fl ow
cells and a fl ow pump (Agilent Technologies, Santa Clara, CA). The dye concentration in
solution was determined by measuring the absorbance at 490 nm. T he amount of the dye
leaching from hair was calculated from the ratio of absorbance to the weight of the hair
tress. The continuous dye release from hair fi bers within 30 min was measured at differ-
ent times with a 2-min interval. Six repetitions per sample were measured. Hair dye
leaching cycles were performed by rinsing with water. Surfactant was not added as it in-
teracts with hair dye and interferes with dye detection.
FT-IR SPECTROSCOPIC IMAGING
A 1-cm-long hair bundle was cut from the middle of the hair tresses and mounted on the
top of a sample holder by embedding in ice. The hair bundle was then microtomed at
-30°C into 5-µm-thick cross sections with a Leica CM 1850 cryostat (Leica Microsystems Inc.,
Bannockburn, IL). Hair cross sections were collected onto CaF2 windows for IR imaging.
This preparation technique avoids any possibility of contamination with embedding or fi xing
medium. Hair cross sections were imaged with a PerkinElmer Spotlight system (PerkinElmer,
Inc., Waltham, MA) that couples a FT-IR spectrometer with an optical microscope. The
system consists of a linear array mercury cadmium telluride detector and an automated
high precision x-y sample stage. Images were acquired with a 6.25 µm step size, eight
scans for each spectrum, and 8 cm-1 spectral resolution. IR spectra were analyzed using
ISys 5.0 software (Malvern Instruments, Inc., Malvern, Worcestershire, UK).
ATOMIC FORCE MICROSCOPY
The hair cross sections were prepared by the following procedure. A single hair fi ber was
hung in the center of a cylinder. Buehlers Epoxicure™ Resin (Buehler, Lake Bluff, IL) and
Buehlers Epoxicure™ Hardener (Buehler) were mixed (weight ratio of 5:1) and slowly
poured into a cylinder. To ensure the hair fi ber remained vertically positioned in epoxy,
one end of the hair fi ber was attached to a thin pole, and the other end was tied with an
JOURNAL OF COSMETIC SCIENCE4
appropriate weight. The epoxy cures slowly at room temperature. After 24 h of curing,
the epoxy containing the embedded hair fi ber was taken out of the cylinder and cut into
~2 mm thick sections perpendicular to the longitudinal axis of the hair fi ber. A standard
metallography polishing technique was to use to polish the epoxy until a clean cross-
sectional interface of hair was obtained. AFM images of the hair cross section were ac-
quired using a Multimode Nanoscope IIId supplied by Bruker Corporation (Billerica,
MA) at ambient conditions (22°C, 50% humidity) in the contact mode. A sharp nitride
lever probe combining a sharp silicon tip with a silicon nitride cantilever was used for the
topographic imaging acquisition. The nominal radius of the tip was about 2 nm and the
spring constant of the cantilever is 0.06 N/m. A scan rate of 2 Hz was used for all mea-
surements. The data collection was set to both the height and defl ection channels.
OPTICAL MICROSCOPY
Images of the hair surface were collected in the refl ected light microscopy mode using an
Olympus BX50 (Olympus America, Center Valley, PA) optical microscope equipped
with 10× (UMPlanFL 10×/0.30), 20× (LMPlanFL 20×/0.40), 50× (LMPlanFL 50×/0.50), and
100× (LMPlanFLN 100×/0.80) objectives. Equipped with a 10× eyepiece and an addi-
tional 2× objective, magnifi cations of roughly 200×, 400×, 1000×, and 2000× were obtained
in the fi nal images. The microscope also contains a motorized z-stage allowing z-stacks to
be generated. Each fi nal image was obtained by generating an image stack and then using
an algorithm to combine in focus details in each image of the stack into one fi nal image.
DYNAMIC CONTACT ANGLE ANALYSIS
An Attension tensiometer was used to determine contact angle (Biolin Scientifi c, Stockholm,
Sweden). A single hair fi ber, cut to approximately 1 cm near the root, was immersed
approximately 3 mm into deionized water and the advancing contact angle was mea-
sured. The required hair diameters were measured using a handheld micrometer. Ten fi bers
in each hair tress were measured.
DIFFERENTIAL SCANNING CALORIMETRY
A Q2000 differential scanning calorimeter (TA Instruments, New Castle, DE) was employed
using pressure resistant, high volume stainless steel pans. Samples consisted of 8–12 mg
of cut (2–5 mm) hair fi bers, along with roughly 55 (±1.5) mg of deionized water. Each
pan was sealed and allowed to sit for at least 6 h at room temperature to ensure equilib-
rium water content and distribution within the hair fi bers. Heating at a rate of 2°C/min
was performed from 22° to 190°C in standard mode. Three repetitions per lot were con-
ducted. TA Universal Analysis 2000 (TA Instruments, New Castle, DE) was used in
conjunction with Windows 4.7A (Microsoft Corporation, Redmond, WA) to determine
the denaturation temperature (Td) and enthalpy of denaturation (ΔH).
RESULTS AND DISCUSSION
To gain more insight into the dye leaching process, we utilized a dissolution apparatus
and monitored the amount of dye leached from hair as a function of rinse time. We studied
both pigmented and nonpigmented hair to understand the contribution of melanin dis-
solution to the dye leaching process. Experiments were carried out using a hair dye based
HAIR DAMAGE DURING MULTIPLE OXIDATIVE DYEING AND SHAMPOOING 5
on pyrazole chemistry, which is often employed in hair dye products designed to provide
red shades. Of the dyes currently used in the market place, the pyrazole dye in this study
provides one of the most intense shades of red, and is especially prone to shampoo or water
removal. Therefore, this dye chemistry is a good choice to better understand the factors
that control color loss.
Several techniques were employed to provide measurements of the physicochemical state
of the hair, focusing on its surface properties (dynamic contact angle analysis) and ultra-
fi ne structure (AFM and refl ected light microscopy). Overall, we fi nd signifi cant damage
in both the surface and internal components of hair due to bleaching and dyeing. Further-
more, dye penetration profi les into the fi ber were monitored with FT-IR spectroscopic
imaging, providing a two-dimensional map across the cross section of hair. And, fi nally,
we followed the condition of hair proteins in the amorphous matrix by measuring Td of
the α-helical, crystalline phase proteins. All of this information allows us to depict a better
picture of how synthetic dyes leach from the hair fi ber.
DYE LEACHING KINETICS OF DIFFERENT TYPES OF HAIR
Figure 1 presents typical kinetics curves corresponding to dye leaching that were acquired
from dyed dark brown and white hair. Dye leaching occurs once dyed hair fi bers come in
contact with water. Hair dye loss rates can be determined from the slopes of the curves.
By calculation, the dye loss rate within the fi rst ~3–5 min is generally 5–8 times faster
than in the later stages, and the dye loss in the fi rst 3 min is ~35% of the total dye loss
(within 30 min). More than likely, the dye loss in the early stages of this process corresponds
to dye migration from the cuticle region to the solution phase since this is the shortest
diffusion route from within the hair structure. Another factor associated with relatively
Figure 1. Plots of leached dye from pigmented and nonpigmented hair fi bers as a function of time. The dye
concentration in solution was determined by measuring the absorbance at 490 nm. The amount of the dye
leaching from hair was calculated from the ratio of the absorbance to the weight of hair tress.
JOURNAL OF COSMETIC SCIENCE6
quick dye loss in the early stages is the high pH in the dyed hair fi ber. Dyeing hair results
in a large increase in pH to ~9. A 2-min rinsing step after the hair dyeing procedure is
not suffi cient enough to lower the pH of hair to 6.5. Under high pH conditions, the cu-
ticles are swelled and therefore provide open channels and pathways for dye to leach out
of the fi bers. It should be noted that most contemporary hair dye systems contain a con-
ditioning step to bring the pH down closer to physiological conditions.
To probe dye spatial distribution inside the hair, dye species were imaged across hair
sections by FT-IR spectroscopic imaging. Figure 2 presents the relative dye concentra-
tion across hair sections. The image was generated by integration ratio of the peak at
1116 cm-1 resulting from the dye species to the amide I peak at 1650 cm-1 due to hair
proteins. Such an analysis indicates that the cuticle region has higher dye concentrations
than the cortex. Free dyes/residues were also visualized in the cuticle areas of the freshly
dyed hair fi ber surface (Figure 3) with an optical microscope. Studies have shown that the
preferred route of reagents to enter the hair fi ber is the scale edge between cuticle cells,
either through the cell membrane complex or endocuticle (11).
During the dyeing process, dye molecules aggregate in the cuticle area as reagents perme-
ate into the hair fi ber. Therefore, the cuticle area is exposed to and contains a higher
amount of dye molecules than other areas of the hair fi ber. These results explain our ob-
servations from the kinetics measurements, which indicate that dye leaching is much
faster in the early stages immediately after dye deposition. Much of the initial dye fading
results from dye loss from the cuticle structure, which is dissolved once the dyed hair fi ber
comes in contact with water. Previous work in this area demonstrated that, in addition to
the cortex, reactions between the dyes and developer also occur at the surface of hair and
in the cuticle (12). In addition, studies by Chandrashekara and Ranganathaiah revealed
that dyes diffuse much more quickly into the cuticle than they do into the cortex (13).
Therefore, it should stand to reason that since a signifi cant quantity of dyes are located in
the cuticle structure, these are likely to diffuse from the hair structure fi rst.
As already noted, most contemporary hair dye protocols involve rinsing, shampooing,
and conditioning steps to bring the pH of hair down closer to physiological conditions
Figure 2. FT-IR image of dye distribution in hair fi ber cross sections. The image was generated by taking
the ratio of the integration of the peak at 1116 cm-1 resulting from the dye species to the amide I peak at
1650 cm-1 resulting from hair proteins.
HAIR DAMAGE DURING MULTIPLE OXIDATIVE DYEING AND SHAMPOOING 7
with the aim to “close” the cuticle and, of course, prevent the leaching of dyes from the
interior of the fi ber. Shampooing plays a major role in the color fastness of hair. In the
experiments conducted in this study only the infl uence of water on hair dye leaching is
considered. It is very likely that results may be different in presence of surfactants.
Dye leaching from dark brown hair is generally faster than from white hair. This trend is
more obvious in the later stages of dye leaching kinetics, which refl ects that dye mole-
cules are transported from the cortex region through the cuticle, and then to the solution.
More than likely, more pores and channels are created in the dark brown hair during the
dyeing process when melanin granules are dissolved. This would provide extra pathways
for dye molecules to leach out. These results are in agreement with previous studies that
demonstrated that hair color fading is greater in pigmented than nonpigmented hair
when exposed to solar radiation in combination with shampooing cycles (14).
INVESTIGATION OF HAIR DAMAGE AND HAIR DYE LOSS DURING MULTIPLE OXIDATIVE
DYEING–LEACHING CYCLES
The hair surface becomes more hydrophilic during the dyeing process. The dyeing proce-
dure removes most lipids from the hair surface since it is generally performed under alka-
line conditions. After the fi rst cycle of the dyeing–leaching process, the contact angle of
hair decreases from 104° for virgin hair to 84° for dyed-leached hair. There is no visible
damage in the internal structure from AFM observations after the fi rst dyeing–leaching
cycle alone. However, if a prebleach process is conducted prior to the hair dyeing process,
Figure 3. A photomicrograph of the freshly dyed hair surface of dark brown hair obtained by refl ectance
light microscopy (scale bar: 10 microns).
JOURNAL OF COSMETIC SCIENCE8
AFM (Figure 4) reveals that the fi ber’s internal structure is severely damaged. Cracks
(over 100 nm in diameter) and holes are observed in the cuticle (e.g., cell membrane
complex, endocuticle) and cortex (e.g., cell membrane complex). Such a result suggests
that the damage caused by the bleaching process facilitates dye loss from the internal
structure of the hair.
To evaluate hair protein damage during the consecutive dyeing–leaching cycles, protein
Td of both dyed European white and dark brown hair were measured after each dyeing
and leaching cycle. The results are displayed in Figure 5. The fi rst three column bars in
each chart demonstrate that Td decreases with consecutive dyeing–leaching cycles, indi-
cating that consecutive dyeing cycles progressively degrade hair proteins. More specifi -
cally, the amorphous matrix becomes more viscous (or plastic) due to damage to its
proteins. It should be noted that Td represents the denaturation temperature of crystalline
phase of the hair; however, changes to the amorphous matrix (which provides support for
the crystalline phase) infl uence the value of Td. Plasticization with water, e.g., shifts Td to
lower temperature, while cross-linking hair (making the amorphous matrix more brittle)
with reactive ingredients increases Td.
The last two column bars in each chart in Figure 5 refl ect how the leaching process alone
affects the hair protein structure. We found that hair dyeing alone affects Td more than
hair dyeing in combination with leaching. Perhaps the leaching process aids in the recov-
ery of hair protein structure. Dyeing the hair results in a large increase in pH to ~9. It
requires extensive rinsing to lower the pH to 6.5. It is possible that when conducting
DSC measurements at high pH the hair protein is in a highly compromised state. These
fi ndings are consistent in both pigmented and nonpigmented hair.
Figure 4. AFM images acquired from hair cross sections in (A) virgin hair and (B) bleached then dyed hair.
The images in (A) and (B) on the left side are of both cuticle and cortex; the images in (A) and (B) in the
middle are from the cuticle region; and the images in (A) and (B) on the right side are from the cortex region.
HAIR DAMAGE DURING MULTIPLE OXIDATIVE DYEING AND SHAMPOOING 9
Interestingly, the dye-leaching rate does not always increase with progressively increasing
amounts of fi ber damage resulting from consecutive dyeing. Figure 6 presents dyeing–
leaching profi les of dyed European dark brown hair after each dyeing–leaching cycle.
After the fi rst dyeing–leaching cycle, we observe an increase in the rate of dye loss. How-
ever, the third, fourth, and fi fth consecutive dyeing–leaching cycles are accompanied by a
decrease in the rate of dye loss. This trend is less noticeable in dyed white hair (data not
shown). These results suggest that deposition of dyes during the initial dyeing steps occurs
in pores and voids formed due to the degradation of melanin and other morphological
components of hair. After the second dyeing process, dye molecules from subsequent dye-
ing steps may granulate with pre-existing dyes already in the fi ber and form larger par-
ticles. The likelihood that large dye particles dissolve slower could explain why the rate
of dye loss decreases on consecutive dyeing–leaching cycles.
CONCLUDING REMARKS
The data generated in this study provide a fundamental understanding of the mecha-
nisms involved in hair dye deposition and dye fastness. Key developments were also made
Figure 5. DSC measurements of multiple dyed and leached hair fi bers for both (A) pigmented and (B) non-
pigmented hair.
JOURNAL OF COSMETIC SCIENCE10
in understanding hair fi ber damage resulting from the hair dyeing process as well as pro-
gressive damage resulting from multiple dyeing cycles. A proprietary sample preparation
technique of hair fi ber cross sections, used in combination with atomic force microscopy,
allowed us to identify key areas of damage within the fi ber including the cell membrane
complex (in both the cuticle and cortex) as well as other regions of the fi ber where we
found small pores and other features consistent with physical damage. More than likely,
these destructed regions provide exit points for existing hair dyes to leach from the fi ber.
Utilizing DSC, we monitored structural changes of keratin protein as a result of hair dye-
ing. In addition to examining the ultrafi ne structure of dyed hair, we also generated a
signifi cant amount of practical data in regard to the kinetics of the leaching process dem-
onstrating differences of leaching in different types of hair and providing real-time data for
this commonly employed consumer procedure. This study confi rms the profound effects
of rinsing/shampooing after dyeing in governing color fastness. Obviously, mitigating the
effects of water and shampoo would help alleviate hair dye loss. As more porous, damaged
hair is more susceptible to color loss, new technologies that minimize damage during
dyeing could facilitate hair dye systems in which the dyes are less prone to undergo leach-
ing during subsequent washing and rinsing steps. Formulators designing color protec-
tion products could use the leaching method to quickly evaluate effects of new ingredients,
shampoos, or hair treatments on dye leaching. The current work also demonstrates that
multiple dyeing processes do not deteriorate hair color loss in spite of progressively dam-
aging the hair fi ber structure. It is speculated that larger granular dye molecules are
formed inside the hair structure during repeated multiple dyeing cycles. Therefore, any
hair dye technologies that facilitate the granulation of dye molecules inside the hair fi ber
should benefi t hair color retention.
Figure 6. Dyeing–leaching profi les from dyed dark brown after each cycle of the dyeing–leaching process.
The dye concentration in solution was determined by measuring the absorbance at 490 nm. The amount of
the dye leaching from hair was calculated from the ratio of the absorbance to the weight of hair tress.
HAIR DAMAGE DURING MULTIPLE OXIDATIVE DYEING AND SHAMPOOING 11
ACKNOWLEDGMENTS
We would like to express our gratitude to Bert Kroon and Linda Foltis of Ashland Spe-
cialty Ingredients for constructive discussions about hair color science.
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