Confocal Raman Spectroscopy of Whole Hairs

Paul D. A. Pudney,a,* Eleanor Y. M. Bonnist,a Kevin J. Mutch,a Rachel Nicholls,a Hugh Rieley,b

Samuel Stanfielda

a Unilever Discover, Colworth Laboratory, Sharnbrook, Bedfordshire, MK44 1LQ UKb Unilever R&D Port Sunlight Laboratory Quarry Road East, Bebington, Wirral, Merseyside, CH63 3JW UK

This paper describes the application of Raman spectroscopy to whole hair

fibers. Previously this has proved difficult because the hairs are relatively

opaque, and spatial resolution diminishes with depth because of the

change in refractive index. A solution is to couple confocal Raman with

multivariate curve resolution (MCR) data analysis, which separates

spectral differences with depth despite this reduction in resolution.

Initially, it is shown that the cuticle can be separated from the cortex,

showing the differences in the proteins, which can then be plotted as a

function of depth, with the cuticle factor being seen only at the surface as

expected. Hairs that had been treated in different ways, e.g., by bleaching,

treatment with the active molecule resorcinol followed by rinsing and

treatment with a full hair care product, were also examined. In all cases,

changes to the hair are identified and are associated with specific parts of

the fiber. Since the hair fiber is kept intact, it can be repeatedly treated

and measured, hence multistep treatment processes can be followed. This

method expands the potential use of Raman spectroscopy in hair research.

Index Headings: Raman; Hair; Keratin; Penetration; Bleaching.

INTRODUCTION

Hair is one of the defining characteristics of mammals. It ismade mainly of protein in a filamentous structure. It has anumber of functions such as providing warmth and camou-flage, as well as for sending signals for danger warnings andmating in some species. In humans many of these functionshave diminished, but it does still provide some function inthermoregulation. The head is the main area where hair is inabundance and where people can relatively easily vary theirappearance. Since people spend a great deal of time and effortcleaning, coloring, and styling their hair, it is of significantimportance to the cosmetic industry.

The structure of the hair fiber has been described elsewhere.1

It consists mainly of the protein keratin in a filamentousstructure and has three main parts: The cortex, whichconstitutes the bulk of the hair, and is formed of microfibrilsand macrofibrils arranged parallel to each other and separatedby a matrix; the cuticle, flattened cells arranged perpendicularto the cortical cells that form the outer protective layer; and themedulla is at the center of the fiber; however, this is often notcontinuous or absent from some human hair fibers. Othercomponents in hair fibers include lipids, water, and melanin,the pigment responsible for hair color.

The constituent protein keratin varies in the order and typesof amino acids present in the different parts of the hair. Theseareas thus have different interactions between groups, i.e.,hydrogen bonds, polar interactions, Coulombic attractions, andcovalent bonds, which lead to different conformations and

secondary structures. For example the cortex material is mainlya-helical, and the cuticle has high b-sheet character.2

Particularly important are disulfide bonds that form betweenthe –SH groups on cysteine residues, which can be intra- orinter-chain and are more prevalent in the cuticle than thecortex.3 These are the principal forces in maintaining thestructure and properties of the hair fiber and are resistant tomany chemical treatments.1

Vibrational spectroscopy is sensitive to molecular structureand conformation of proteins4 and so is well placed toinvestigate the properties of hair. Indeed there are a number ofstudies using both infrared (IR) and Raman spectrosco-py5,6,7,8,9,10,11 and also coherent anti-Stokes Raman scatteringspectroscopy (CARS).12,13 When cross-sectioned, the differentstructures of the cuticle and cortex have been demonstrated bythese methods.2,6,14 Changes in hairs have also been followedwhen treated in various ways, the most widely used treatmentbeing bleaching.3,13,15 A treatment of hair with cosmeticproducts often involves diffusion of active compounds intohair.16 The penetration of actives has been previously shownby Raman and IR.9,17

Despite much progress having been made, some challengesstill remain, which in combination prevent valuable insightsinto hair properties and hair treatment from being uncovered.Most studies have concerned microtomed hair rather thanwhole hair fibers, allowing access to the different areas of thefiber by scanning along the cross-section. The drawback to thisis that often one may want to follow external moleculepenetration over time or examine the hair throughout asuccessive treatment regime. Microtoming destroys the sampleso that this type of repeat measurement is not possible and alsonegates one of the advantages of Raman spectroscopy of beingnoninvasive. This approach is taken because, when samplingwhole hair, especially when doing depth profiles through wholehair fibers, it is difficult to distinguish clearly the differentlayers of the hair structure.18 This is because the hair structureis composed predominantly of keratin, and it is only relativelyminor inhomogeneities in its chemical and structural compo-sition that distinguish different layers, as highlighted above.The difficulty in unambiguously detecting and mapping theselayers is further compounded by the fact that the Ramanspectroscopic signals of different proteins and their differentstructural conformations are all very similar and heavilyoverlap. Furthermore, the natural optical properties of hairleads to a large change in refractive index, ongoing from airinto the hair fiber, which causes the focus of the Raman laserbeam to be enlarged, thus reducing the confocal resolution withincreasing depth and condensing of the depth scale (theapparent depth problem).19 Recently, Zhang et al.20 made useof the normal method of reducing the loss of depth resolution19

by using an oil-immersion objective in order to refractive-indexmatch the sample. They did this by placing the hair under a

Received 27 March 2013; accepted 3 August 2013.* Author to whom correspondence should be sent. E-mail: paul.pudney@unilever.com.DOI: 10.1366/13-07086

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� 2013 Society for Applied Spectroscopy

coverslip to prevent oil from interfering with the hair. Thisprovided a good depth profile and showed protein featureschanging with depth, although spectral overlap was stillapparent when compared with the separated cuticle and cortexspectra2 and when microtomed.6,13,14,21 They did not comparedata collected using immersion and dry objectives.

Hence, there is a need for an improved methodology that canaccommodate the measurement of whole hair fibers andsufficiently discriminate between the different sections of thehair within the data that is generated. Essentially whole hairsare a multicomponent microstructure, and thus a Raman mapproduces a large spectral dataset. In a conventional analysis ofthe resultant spectrum, an attempt is made to attribute specificindividual spectral intensities to specific chemical componentsand then derive, as a function of position, the spatialcomponent maps. For the spectra generated by a multicompo-nent microstructure, this is generally not possible since it israrely possible to unambiguously attribute specific spectralintensities in this way. This is clearly the case in spectra of hairas described in the above section, as it comprises mainlykeratin but differently folded. Consequently, a differentapproach is needed, in which the aim of the analysis is toproduce pure separated spectra and their intensities from thespectral dataset. In a lot of chemometric techniques, the spectraare separated on the basis of variations, in this case in thespatial and spectral dimensions. However, in most methods,this does not produce pure and thus interpretable factors.However, there are methods for doing this, which generally

come under the title of self-modeling curve resolution (SMCR)or multivariate curve resolution (MCR).22

This method has now been successfully used for a number ofyears on different types of microstructures that have beenRaman-mapped. These include emulsions,23 phase separatedbiopolymers,24 food products,25,26 and plant structures.27 It hasalso been used on depth profiles into skin,28 which is mostsimilar to the system being examined in this study.

This paper reports the spectral separation of the differentparts of hair fibers from depth-profile maps of whole hairs withmultivariate curve resolution (MCR) analysis. This is demon-strated with yak hair (a good model of human hair withoutmelanin) and blonde or gray human hair. The method is furtherapplied to a number of hair problems, specifically to investigatebleaching and external molecule treatment without microtom-ing the hair sample. It is found that bleaching mainly affects thecuticle layer, and its extent diminishes with depth. We showthe uptake of resorcinol into the hair fiber and observe that theamount inside the hair is reduced on rinsing. Finally, weidentify the penetration of a select ingredient into the hair fiberafter treatment with a full hair-care product formulation. Theuse of air objectives allows the most flexibility with treatmentconditions, but the approach would be equally applicable todata collected with immersion objectives in those cases wherethe sample allows it. This approach expands the applicability ofRaman in applied hair research.

METHODS

Hair Samples. Yak hair was obtained from Hugo RoyerLtd. as premade 0.75 g, 5 cm length switches of yak belly hair,which is a close approximation to human hair. It is very low incolor and is usually obtained with little damage and is ofconsistent quality. Natural white hair was obtained fromInternational Hair Importers, USA; selected by opticalmicroscopy were naturally blonde virgin human hair.

To bleach the hair strands, L’Oreal Platine Precision bleachpowder and 9% Excel creme peroxide were mixed in a 1 : 2ratio. The bleaching mix was applied to each hair strand using atinting brush and left to develop for 30 min. Each hair strandwas rinsed for 2 min and left to air dry for 2 h. Following this,another bleach powder/peroxide creme mixture was preparedand applied once more to the bleached hairs. After leaving for30 min, the hair strands were washed using a base shampoo(17.10 wt % sodium laureth sulfate, 5.33 wt % cocoamido-propyl betaine,0.40 wt % DMDM hydantoin, 1.40 wt %sodium chloride, in water) and air-dried.

FIG. 1. Schematic diagram showing how the whole hair fibers were sampled.Spectra are collected along the length of the hair with a depth profile collectedat each point.

FIG. 2. The two main factors/spectra obtained from mapping a virgin yak’s hair using the Kaiser holoprobe 5000R spectrometer. (a)The blue spectrum is typical ofkeratin with high a-helix content, typical of the cortex. (b) The red spectrum, shows an S-S rich/b-sheet protein of the cuticle.

APPLIED SPECTROSCOPY 1409

The procedure for treating yak hair with resorcinol (1,3-dihydroxy benzene) was as follows: 0.4 g of 15 cm long yakhair was packed into a glass pipette. This was then rinsed with1 mL of a 2% by weight solution of resorcinol. For the rinsesample, the treated hair was left for 24 h then rinsed with 2 mLof distilled water.

Blonde human hairs were treated with a full formulationNeXXus product, Youth Renewal, whereby the 1% glycerol inthe formulation was replaced with deuterated glycerol. This is aleave-on formulation, so hairs were treated with the productand left for approximately 24 h before measurement.

Data Collection. Raman spectra were collected usingidentical procedures on two different spectrometers, both usingan excitation wavelength of 785 nm. The first was a WITecAlpha 300 R system. The 785 nm laser was used at an operatingpower of 50 mW before the objective; this was selected such asto avoid sample damage while still resulting in sufficient sampleexcitation. A Zeiss 1003 (N.A. 0.9) air/metallurgical objectiveand a Zeiss Achromoplan 1003 (N.A. 1.25) immersion objectivewere used. The second spectrometer used was a Kaiserholoprobe 5000R confocal Raman spectrometer equipped withan Olympus air/1003 metallurgical objective (N.A. 0.8), andalso employing less than 50 mW of laser power at the sample.

Hair strands were fixed to a glass microscope slide usingtape, ensuring that the hair fiber was pulled straight withoutstretching. When using the oil-immersion objective, a coverslip was placed on top of the hair so the immersion oil did notinterfere with the hair. A line depth profile along the length ofthe hair was collected, i.e., an xz-map of the hair fiber, as

shown in Fig. 1. The collection of data along the length of thefiber, rather than across the fiber, prevents some more seriousproblems concerning the mixing of spatial information.29

A collection time of 60 s for each point was used with bothspectrometers unless otherwise stated.

Data Analysis. Multivariate curve resolution (MCR)analysis separates the data into two modes: one whichdescribes the spectra of the different components present (thefactors) and another describing the intensity of each spectralfactor, at each location sampled (the scores)22. To do this, aprincipal factor analysis (PFA), or equivalent method, isperformed on the unscaled data. The factors represent thechanges which occur in the spectra and are calculated byseparating them out according to their variance from eachother, i.e., as different from each other as possible. A relativescore is given to each factor to show the distribution of thesefactors through the sample. After PFA, the resulting factors arenot easily interpreted as they may still be mixtures of thecomponents and often do not represent the real components.30

Further constraints must be applied to transform the results intoreal spectra and pure scores; these constraints are applied usingan alternating least squares regression (ALS) algorithm.29 Anon-negativity constraint is applied to make the data practicallymeaningful, i.e., it is not possible to have a negativeconcentration (score) or negative Raman peaks. An equalityconstraint is also applied; this aims to normalize the factorsensuring that all intensity variance is found in the scores andnot in the spectra.30 The constraints are applied alternatively tothe factors and scores until a solution converges, thus

FIG. 3. A plot of the two main factors obtained from MCR analysis of a xz scan through a single yak hair fiber. The y-axis is a line of points along the top of the hairat 2 lm intervals (x-scan), and the x-axis is the z direction, i.e., penetrating through the hair, with points taken at 5 mm intervals. A collection time of 60 s was used ateach point.

FIG. 4. Spectral factors from MCR analysis of the data from mapping an untreated whole human hair fiber using the WITec Alpha 300 R spectrometer. Blue is thecortex. Green is the cuticle. For label meanings see text.

1410 Volume 67, Number 12, 2013

producing factors representing the real spectra of thecomponents present and scores that represent the relativeconcentration of each factor at every position sampled. This isthe basic ALS approach; the modified alternating least squares

(MALS) algorithm is an improved implementation of thismethod and has been described elsewhere.31

When conducting a depth profile, one of the main problems isthe loss of ongoing signal deeper into the sample, mostly causedby physical scattering and spherical aberration. The sameproblems are encountered when doing depth profiles intoskin.32,33 This problem is overcome by using the protein signalas an internal standard as the concentration of protein isapproximately constant with depth. Here, the same approach canbe taken using the keratin signal. Another problem withmeasuring depth is that the depth axis is not real depth intothe sample but rather apparent depth; the depth axis here refersto mechanical depth. This is the distance moved by theobjective from the surface position of the hair fiber to collect thespectrum at that point.

RESULTS AND DISCUSSION

Whole Untreated Hairs. Spectra were collected in a linealong the length of the hair taking a depth profile at each point(an xz-map, see Fig. 1). Firstly, yak hair was examined. Thisxz-map data was then analyzed using the MCR method; the twomain spectral factors resulting from this are shown in Fig. 2.The associated scores (intensities) are plotted in Fig. 3. Thisexperiment has been repeated on human whole blonde hairs,and the same analysis carried out; the spectral factors arecomparable and shown in Fig. 4. The associated score maps arevery similar to those of yak hair that are shown in Fig. 3, so arenot shown here. From the spatial distribution and the featuresof the spectral factors, these can be associated with the cuticle(Figs. 2b and 4 green) and cortex (Figs. 2a and 4 blue). Thespectral factors highlight the differences between the twoproteins in both molecular structure and conformation; the keydifferences and peak assignments5,14,34 are listed in Table 1.Here, the features from the human hair are discussed, but thisapplies equally for the yak hair results as well.

TABLE I. Assignments of cuticle and cortex spectrum derived from thespectral factors produced from MCR analysis. Assignments usingreferences 2, 3, 5, 14, and 36.

Cuticle/cm�1 Cortex/cm�1

v (SS) (gauche–gauche–gauche) 509 509

v (CS) (gauche) 644, 663, 671 –v (CS) 686, 719 –p (CH2) in-phase – 742/743d (CCH) aliphatic (tyrosine

side chains) – 828d (CCH) aromatic (tyrosine

side chains) – 853p (CH2) tryptophan 880–891 –v (CC) helix-a/p (CH3)

terminal – 933p (CH3) d (CCH) olefinic – 959- 982, 989 –v (CC) aromatic ring/

phenylalanine – 1002-1003v (CC) skeletal/cis

conformation 1033 –v (CC) skeletal/trans

conformation 1044 1047v (CC) skeletal 1052 –

v (CC) skeletal –1082�1103

and 1125Tyrosine 1176, 1187, 1199 –Amide III disordered 1241, 1252 –Amide III 1281 –Amide III a helix – 1342CH2 bending mode 1451, 1465 1448

Amide I1675

(1668–1679)1657

(1649–1669)

FIG. 5. Plots of the scores of the cortex and cuticle from MCR analysis of xz depth profiles from the same untreated yak’s hair. (a) A plot of the cortex scores usingthe air objective. (b) The plot of cuticle scores using the air objective. (c) A plot of the cortex scores using the oil objective. (d) The plot of cuticle scores using the oilobjective. Data collected using WITec Alpha 300 R spectrometer.

APPLIED SPECTROSCOPY 1411

The spectra reflect differences in amino acid content,specifically that the cuticle is more sulfur-rich than the cortexdue to a higher number of cysteine residues14 and that thecortex has more tyrosine and phenylalanine.2 The band at 509cm�1 in Fig. 4a, which arises from the disulfide (-C-S-S-C-)bonds,35 is more intense in the cuticle spectrum. The narrowershape of this band in the cuticle, suggests that the SS bondshere have a high population of the gauche-gauche-gaucheconformation. An all-gauche conformer is centered at ;510cm�1, but this increases in frequency when a trans conforma-tion is introduced (these conformers are centered at 525, 540cm�1). The higher intensity of the 645 cm�1 band for thecuticle, Fig. 4b, from the C-S bonds, is also consistent withhigher levels of cysteine. The bands at 828 and 853 cm�1, Fig.4c, are only evident in the cortex spectrum; these originatefrom the amino acid tyrosine. The phenylalanine peak at;1002 cm�1 in Fig. 4e is more intense in the cortex spectrum,also fitting with the known amino acid content.

Apart from reflecting the different amino acids, the resolvedcuticle and cortex spectra further show the differences due totheir conformations, the cuticle being in the b-sheet confor-mation, while the cortex is a-helical. This can be seen from theposition of the amide I band, Fig. 4g, which is centered at 1675cm�1 in the cuticle and at 1657 cm�1 for the cortex, and of theamide III band, Fig. 4f, which is between 1240 and 1252 cm�1

for the cuticle, and at 1342 cm�1 for the cortex. In addition to

these are the presence of a band at 933 cm�1 in the cortex andthe absence of a 959 cm�1 band in the cuticle.

These spectral factor results for cuticle and cortex are ingood agreement with Raman spectra from the same partsisolated from sheep’s hair (i.e., merino wool).2 Spectra fromthe cuticle and cortex of hair have been identified from a cross-section of hair;14,36 however, the differences observed here areless well defined and often contain some features of the otherproteins present. The spectra obtained on a whole hair using anoil objective showed differences with depth consistent with thechanges outlined above, including a shift of the amide I bandtowards the a-helical position and the lower intensity of the S-Sband;20 however, the features are not as well separated as seenusing the MCR analysis.

The plot of the scores of these two spectral factors in Fig. 3show that the cuticle factor is only observed at the surface ofthe hair and then appears again when the focus reaches theother side of the hair; although, at this stage, the laser focus isvery diffuse, and hence here, it appears much more spatiallyspread. The cortex factor appears between the location of thecuticle factor, as would be expected.

Thus, application of the MCR analysis method has provideda clear resolution of cuticle and the cortex layers in the datafrom a whole hair fiber, without the need for microtoming. Theintact sample is thus available for further measurement, givingscope for continued investigation.

Comparing Collection Methods. The use of dry objectivesallows the most flexibility with treatment conditions; however,as pointed out in many publications,19,37,38 this leads todegraded resolution in the z-axis. The normal way to negatethis is to use immersion objectives to refractive index match.This approach has been used with hair20 where a coverslip isplaced on top of the hair. Here, this method is compared withthe dry objective for hair. Data was collected from the sameyak’s hair using the same collection conditions for the airobjective but with an oil-immersion objective and coverslip.This data was analyzed by the MCR methods and results invery similar factors, to those shown in Figs. 2 and 4. The scoremaps showing the spatial distribution are shown in Fig. 5. Bothmethods clearly differentiate the cuticle and cortex spatially;

FIG. 6. Spectral factors from untreated (blue) and bleached blonde human hair (red). (a) Cuticle factors. (b) Cortex factors. Data collected using WITec Alpha 300R spectrometer.

FIG. 7. Ratio of the sulfur-oxygen peak (1020–1070 cm�1) area tophenylalanine peak (990–1016 cm�1) area on the same hair before (red) andafter bleaching (blue). The five points along each hair have then been averagedto give a representative ratio for the hairs at each treatment stage. Data collectedusing WITec Alpha 300 R spectrometer.

1412 Volume 67, Number 12, 2013

the main difference is that the signal from the cortex ismaintained to greater depth with the oil-immersion objective asthe laser focus is maintained much better, whereas the focusthrough the dry objective is much more attenuated because ofthe larger refraction effect.38 However, the dry-objective datagives a good signal from the cortex well beyond the cuticleregion, suggesting that the cortex can be measured with goodreliability.

One practical issue that should be mentioned is that goodprofiles are not always obtained when collecting data using theoil objective because, as the hair fiber is not flat, being bothround and rough, the coverslip is not always in good contactwith the hair over a large area.

Effect of Bleaching on Hair. Whole blonde human hairswere examined before and after bleaching. The data was thenanalyzed using the MCR method. The cuticle and cortexspectral factors from this analysis are compared before andafter the bleaching process, as shown in Fig. 6. The bleachedcuticle shows a reduction in the S-S band at 508 cm�1 and anincrease in the S-O band seen at ;1042 cm�1. These changesare also replicated in the cortex factors, see Fig. 6b, but theincrease in the S-O band is larger in the cuticle than in thecortex. Thus, the impact of bleaching spans across differentlayers of the hair fiber. To show this in further detail, a plot ofthe integrated intensity ratio of the sulfur-oxygen band (1020–1070 cm�1) normalized to the phenyalanine band (990–1016cm�1) with depth is shown for the same hair before and afterbleaching, see Fig. 7. This further illustrates that the S-O bandincreases upon bleaching across the measured depth range andthat the extent of this change decreases with depth.

The spectral differences observed here are consistent withthe molecular changes known to be caused by bleaching,namely cleavage and oxidation of the disulfide bonds tosulfonic acid (conversion of cystein to cysteic acid39). Anumber of other studies have investigated bleaching,3,14,15,39

and most are in agreement. The impact is greatest in thecuticle14,39 and diminishes in the deeper layers of the fiber, as isexpected from a surface insult. Multivariate curve resolutionanalysis has enabled specific examination of the cuticle andcortex layers separately from a completely intact hair.Furthermore, variability in the data has been reduced bymeasuring the same hair fiber before and after bleaching.

Commercially, hair is bleached for cosmetic reasons tolighten the color of hair, either as the sole aim or as a base forwhich the hair can then be colored. Bleaching decolorizes thehair strand by breaking down the melanin pigment but alsodamages the protein structure, breaking apart the disulfidebonds, which leads to swelling of the fiber, brittleness, anddryness. The ideal bleaching process would break down themelanin without affecting the keratin structure. Consequently,being able to follow the bleaching process on whole hairs, itsextent, and where any changes occur spatially is a valuablecapability for the investigation of existing hair treatments andfor the search for new treatments that are more selective.

Penetration into Hairs. Resorcinol. Yak hair was treatedwith a solution of resorcinol (1,3-dihydroxy benzene), andRaman line depth profiles were collected on both treated anduntreated hairs. The MCR analysis of the dataset resolves aspectral factor that closely resembles the Raman spectrum ofthis molecule, shown in Fig. 8. Figure 9 shows the spatialdistribution plots from the treated hair map. Figure 9a is a mapof the keratin intensity, which shows the location of the hairsubstrate; Fig. 9b shows the corresponding resorcinol intensity;and Fig. 9c shows the resorcinol normalized to the keratincomponent. Together, these illustrate that the resorcinol haspenetrated extensively, is widely distributed through the hairfiber, and has an approximately uniform concentrationthroughout.

Figure 10 shows the data obtained from hair that has beenrinsed after the treatment with resorcinol. Resorcinol was stillpresent within the hair and remained evenly distributed. Thedata from both maps were analyzed together with the MCR as asingle combined dataset, and so the score values calculated for

FIG. 8. Spectral factor for resorcinol (blue) compared to spectrum of pureresorcinol (red). Data collected using Kaiser holoprobe 5000R spectrometer.

FIG. 9. Spatial distribution maps of yak’s hair treated with resorcinol. (a) Keratin factor. Showing the position of the hair, (b) map of resorcinol. (c) Normalizedresorcinol factor. Data collected using Kaiser holoprobe 5000R spectrometer.

APPLIED SPECTROSCOPY 1413

the resorcinol in each are directly comparable. It shows 60% ofthe resorcinol initially present on application is retainedfollowing rinsing. In a separate chemical analysis the percentretention of resorcinol was measured by using dry weightanalysis to quantify the uptake of bulk hair fibers from an initial2% w/w solution and the amount of resorcinol collected in thewater eluent used to rinse the same fibers. This method alsomeasured 60% retention for resorcinol to compare favorablywith the results from the Raman study.

Penetration of actives has been previously measured withRaman and IR9,17 on microtomed hair samples. But here, wehave shown that it is possible to do this with whole hairs andthis gives the benefit of being able to follow the same samplethrough a treatment regime, without affecting the underlyingsubstrate by invasive analysis.

Glycerol from a Full Product. Understanding delivery froma full product, given the many components that are normallypresent, is a bigger challenge than a single compound insolution. Clearly, it is also the crucial test of whether a productis doing what it is expected to do, as other ingredients mayinterfere. In principle, this is possible using an MCR analysis

approach, as has been shown for a full food product.26

However, a molecule may only make up a small percentage ofthe product, and then multiple band overlap may become toomuch of a problem and require acquisition of a very largedataset. One way to overcome this or reduce the size of theneeded dataset is to use a deuterated version of the molecule ofinterest, as has been used in skin penetration, for example.40

This is the approach taken here with glycerol. The product(NeXXus Youth Renewal) contained 1% glycerol, which wasreplaced with d-glycerol. The treated hairs (see Methodssection) were then mapped as previously described, but withlonger acquisition times, 4 min per spectrum. A plot of thelocation of glycerol can be done using the intensity of the C–Dstretch alone. However, we have again used the MCR method,as this allows better separation of other components, especiallythe keratin, which is then used to normalize the glycerolintensity inside the hair (see Methods section) as deuteratedglycerol still has bands overlapping many of the protein bands,see Fig. 11. Using MCR overcomes these backgroundinterference problems. A plot of the glycerol location is shownin Fig. 12. It can be seen that the glycerol clearly penetrates thehair cortex, which is an important observation to helpunderstand and exploit the potential benefits of this agentwhen applied as part of a hair product.

CONCLUSION

The use of confocal Raman spectroscopy has beendemonstrated on whole hairs, and it has been shown that, withthe right analysis methods, the different sections of the hair(cuticle and cortex) can be differentiated both spectroscopicallyand spatially, despite the diminished resolution from refractiveindex changes when scanning into the hair.19 This method wasapplied to investigations on hair treatment, using whole hairfibers for measurement. Cuticle and cortex were affected todifferent extents in hairs that had been bleached. Penetration of

FIG. 10. Comparison of resorcinol factors intensity maps before and after rinsing of the hair. Data collected using Kaiser holoprobe 5000R spectrometer.

FIG. 11. (a) Spectral factors for the hair fiber (keratin) and (b) the d-glycerolfrom the MCR of a hair treated with shampoo product containing 1% d-glycerol. (c) The Raman spectrum of d-glycerol. Data collected using WITecAlpha 300 R spectrometer.

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a target molecule was identified, from both a solution and a fullproduct formulation, which reveals whether a molecule hasreached its intended target. It was also shown that a treatmentcycle could be followed, in this case rinsing. This demonstratesthat Raman spectroscopy is in a position to be more widelyapplied to hair research with a key role to play in the selectionof new benefit agents based on their physical, chemical, andspatial efficacy.

ACKNOWLEDGMENTS

Eleanor D’Agostino, Ranjit Bhogal, Glynn Roberts, Richard Read, GezAdams, Janhavi Raut, Cheryl Taylor, Michael Brown.

1. C.R. Roberts. Chemical and Physical Behavior of Human Hair. New York:Springer-Verlag, 2002. 4th ed.

2. J.S. Church, G.L. Corino, A.L. Woodhead. ‘‘The Analysis of Merino WoolCuticle and Cortical Cells by Fourier Transform Raman Spectroscopy’’.Biopolymers. 1997. 42(1): 7-17.

3. W. Akhtar, H.G.M. Edwards, D.W. Farwell, M. Nutbrown. ‘‘Fourier-Transform Raman Spectroscopic Study of Human Hair’’. Spectrochim.Acta, Part A. 1997. 53(7): 1021-1031.

4. A. Barth, C. Zscherp. ‘‘What Vibrations Tell Us About Proteins’’. Q. Rev.Biophys. 2002. 35(4): 369-430.

5. A.C. Williams, H.G.M. Edwards, B.W. Barry. ‘‘Raman Spectra of HumanKeratotic Biopolymers: Skin, Callus, Hair and Nail’’. J. Raman Spectrosc.1994. 25(1): 95-98.

6. A. Kuzuhara, N. Fujiwara, T. Hori. ‘‘Analysis of Internal StructureChanges in Black Human Hair Keratin Fibers with Aging Using RamanSpectroscopy’’. Biopolymers. 2007. 87(2–3): 134-140.

7. A. Kuzuhara. ‘‘Protein Structural Changes in Keratin Fibers Induced byChemical Modification Using 2-Iminothiolane Hydrochloride: A RamanSpectroscopic Investigation’’. Biopolymers. 2005. 79(4): 173-184.

8. K.L.A. Chan, S.G. Kazarian, A. Mavraki, D.R. Williams. ‘‘FourierTransform Infrared Imaging of Human Hair with a High Spatial ResolutionWithout the Use of a Synchrotron’’. Applied Spec. 2005. 59(2): 149-155.

9. K.L.A. Chan, F.H. Tay, C. Taylor, S.G. Kazarian. ‘‘A Novel Approach forStudy of In Situ Diffusion in Human Hair Using Fourier TransformInfrared Spectroscopic Imaging’’. Applied Spec. 2008. 62(9): 1041-44.

10. K.R. Ackermann, J. Koster, S. Schluecker. ‘‘Polarized Raman Microspec-

troscopy on Intact Human Hair’’. J. Biophotonics. 2008. 1(5): 419-424.

11. F.I. Bell, R. Skinner. I.M. Tucker, L. Leray, T.E. Lyons, K. Devine P.

Pudney, T. Oikawa. ‘‘Biophysical and Mechanical Response of Keratinous

Fibres to Changes in Temperature and Humidity’’. J. Cosmet. Sci. 2004.

55(Suppl.): S19-S24.

12. M. Zimmerley, C.-Y. Lin, D.C. Oertel, J.M. Marsh, J.L. Ward, E.O.

Potma. ‘‘Quantitative Detection Of Chemical Compounds In Human Hair

With Coherent Anti-Stokes Raman Scattering Microscopy’’. J. Biomed.

Opt. 2009. 14(4): 044019.

13. K. Bito, M. Okuno, H. Kano, S. Tokuhara, S. Naito, Y. Masukawa, P.

Leproux, V. Couderc. and H.-O. Hamaguchi. ‘‘Protein Secondary Structure

Imaging with Ultrabroadband Multiplex Coherent Anti-Stokes Raman

Scattering (CARS) Microspectroscopy’’. J. Phys. Chem. B. 2012. 116(4):

1452-1457.

14. A. Kuzuhara. ‘‘Analysis of Structural Changes in Bleached Keratin Fibers

(Black and White Human Hair) Using Raman Spectroscopy’’. Biopoly-

mers. 2006. 81(6): 506-514.

15. L.J. Hogg, H.G.M. Edwards, D.W. Farwell, A.T. Peters. ‘‘FT Raman-

Spectroscopic Studies Of Wool’’. J. Soc. Dyers Colour. 1994. 110(5–6):

196-199.

16. L. Wang, L. Chen, L. Han, G. Lian. ‘‘Kinetics and Equilibrium of Solute

Diffusion into Human Hair’’. Ann. Biomed. Eng. 2012. 40: 2719-2726.

17. A. Kuzuhara. ‘‘Raman Spectroscopic Analysis of L-Phenylalanine and

Hydrolyzed Eggwhite Protein Penetration into Keratin Fibers’’. J. Appl.

Polym. Sci. 2011. 122(4): 2680-2689.

18. L.E. Jurdana, K.P. Ghiggino, K.W. Nugent, I.H. Lever. ‘‘Confocal Laser

Raman Microprobe Studies of Keratin Fibers’’. Text. Res. J. 1995. 65(10):

593-600.

19. N.J. Everall. ‘‘Confocal Raman Microscopy: Performance, Pitfalls, and

Best Practice’’. Appl. Spectrosc. 2009. 63(9): 245A-262A.

20. G. Zhang, L. Senak, D.J. Moore. ‘‘Measuring Changes in Chemistry,

Composition, and Molecular Structure Within Hair Fibers by Infrared

and Raman Spectroscopic Imaging’’. J. Biomed. Opt. 2011. 16(5):

056009.

21. A. Kuzuhara. ‘‘Analysis of Structural Changes in Permanent Waved

Human Hair Using Raman Spectroscopy’’. Biopolymers. 2007. 85(3): 274-

283.

22. P.D.A. Pudney, T.M. Hancewicz. ‘‘The Role of Confocal Raman

Spectroscopy in Food Science’’. Handb. Vib. Spectrosc. 2010:133-167.

FIG. 12. (a) Score maps for the hair fiber (keratin) and (b) the d-glycerol normalized to hair from the MCR analysis of a hair treated with shampoo productcontaining 1% d-glycerol. Data collected using WITec Alpha 300 R spectrometer.

APPLIED SPECTROSCOPY 1415

23. J.J. Andrew, M.A. Browne, I.E. Clark, T.M. Hancewicz, A.J. Millichope.‘‘Raman Imaging of Emulsion Systems’’. Appl. Spectrosc. 1998. 52: 790-796.

24. P.D.A. Pudney, T.M. Hancewicz, D.G. Cunningham, C Gray. ‘‘A NovelMethod for Measuring Concentrations of Phase Separated Biopolymers:The Use of Confocal Raman Spectroscopy with Self-Modelling CurveResolution’’. Food Hydrocolloids. 2003. 17(3): 345-353.

25. P.D.A. Pudney, T.M. Hancewicz, D.G. Cunningham. ‘‘The Use ofConfocal Raman Spectroscopy to Characterise the Microstructure ofComplex Biomaterials: Foods’’. Spectrosc. 2002. 16(2-3): 217-225.

26. P.D.A. Pudney, T.M. Hancewicz, D.G. Cunningham, M.C. Brown.‘‘Quantifying the Microstructures of Soft Solid Materials by ConfocalRaman Spectroscopy’’. Vib. Spectrosc. 2004. 34(1): 123-135.

27. P.D.A. Pudney, L. Gambelli, M.J. Gidley. ‘‘Confocal Raman Microspec-troscopic Study of the Molecular Status of Carotenoids in Tomato Fruitsand Foods’’. Appl. Spectrosc. 2011. 65(2): 127-134.

28. E.Y.M. Bonnist, J.-P. Gorce, C. Mackay, R.U. Pendlington, P.D.A.Pudney. ‘‘Measuring the Penetration of a Skin Sensitizer and Its DeliveryVehicles Simultaneously with Confocal Raman Spectroscopy’’. SkinPharmacol. Physiol. 2011. 24(5): 274-283.

29. N.J. Everall. ‘‘Otimising Image Fidelity in 3D Confocal Raman Imaging’’.Paper presented at: SciX Conference. Kansas City, MO; Sept 30-Oct. 52012.

30. J.J. Andrew, T.M. Hancewicz. ‘‘Rapid Analysis of Raman Image DataUsing Two-Way Multivariate Curve Resolution’’. Appl. Spectrosc. 1998.52(6): 797-807.

31. J. Wang, P.K. Hopke, T.M. Hancewicz, S.L. Zhang. ‘‘Application ofModified Alternating Least Squares Regression to Spectroscopic ImageAnalysis’’. Anal. Chim. Acta. 2003. 476(1): 93-109.

32. P.D.A. Pudney, M.E. Melot, P.J. Caspers, A. Van Der Pol, G.J. Puppels.

‘‘An In Vivo Confocal Raman Study of the Delivery of Trans Retinol to the

Skin’’. Appl. Spectrosc. 2007. 61(8): 804-811.

33. M. Melot, P.D.A. Pudney, A.-M. Williamson, P.J. Caspers, A. Van Der

Pol, G.J. Puppels. ‘‘Studying the Effectiveness of Penetration Enhancers to

Deliver Retinol through the Stratum Cornum by In-Vivo Confocal Raman

Spectroscopy’’. J. Controlled Release. 2009. 138(1): 32-39.

34. R. Paquin, P. Colomban. ‘‘Nanomechanics of Single Keratin Fibres: A

Raman Study of the a-Helix ! b-Sheet Transition and the Effect of

Water’’. J. Raman Spectrosc. 2007. 38(5): 504-514.

35. M. Gniadecka, O.F. Nielsen, D.H. Christensen, H.C. Wulf. ‘‘Structure of

Water, Proteins, and Lipids in Intact Human Skin, Hair, and Nail’’. J.

Invest. Dermatol. 1998. 110(4): 393-398.

36. A. Kuzuhara, T. Hori. ‘‘Reduction Mechanism of L-cysteine on Keratin

Fibers Using Microspectrophotometry and Raman Spectroscopy’’. Bio-

polymers. 2005. 79(6): 324-334.

37. N.J. Everall. ‘‘Confocal Raman Microscopy: Why the Depth Resolution

and Spatial Accuracy Can Be Much Worse Than You Think’’. Appl.

Spectrosc. 2000. 54(10): 1515-1520.

38. N.J. Everall. ‘‘Modeling and Measuring the Effect of Refraction on the

Depth Resolution of Confocal Raman Microscopy’’. Appl. Spectrosc.

2000. 54(6): 773-782.

39. L.J. Wolfram, K. Hall, I. Hui. ‘‘The Mechanism of Hair Bleaching’’. J. Soc.

Cosmet. Chem. 1970. 21: 875-900.

40. G. Mao, C.R. Flach, R. Mendelsohn, R.M. Walters. ‘‘Imaging the

Distribution of Sodium Dodecyl Sulfate in Skin by Confocal Raman and

Infrared Microspectroscopy’’. Pharm. Res. 2012. 29(8): 2189-2201.

1416 Volume 67, Number 12, 2013