Tech Edge: Optical Tools to Examine the Structure of Cosmetic Products

From the column editor: Within the past five years, the timeline for a formulation to go from the bench to market has shortened significantly. Today, every chemist is faced with creating a new, stable formula within months when it used to take over a year. To help chemists measure the stability of formulas within a reasonable time frame, accelerated stability testing is used; however, this still requires at least three months of testing. Many companies are looking for ways to examine their formulations to attempt to relate physical structure to stability.

There are a number of new tools that have entered the marketplace and are finding use in physical sciences. These tools can be used in the laboratory to analyze the structure of a product. In this month’s column, Phil Cummins, PhD, of Estée Lauder, discusses old and new microscopic techniques that can be used to analyze product structure. Many of these non-invasive techniques are also used to look at skin structure in vivo for claims support.

Cummins has worked in various areas of measurement and material science, including forms of scattering and spectroscopy—especially with their relevance to surface and colloid properties of soft solids. He received his doctoratal degree in chemical physics from the University of Sheffield, UK, and gained postdoctoral experience at the University of Southern California and Massachusetts Institute of Technology before returning to the UK to work for Unilever.

At Unilever, he rose to senior manager for measurement sciences and was responsible for long term measurement research for the company as well as management control over analytical sciences at the Port Sunlight Laboratory. In 2002, after a long research career with Unilever, UK, Cummins joined Estée Lauder as executive director for measurement and material science.

A cosmetic product is a complex, nonequilibrium mixture of ingredients including polymers, small molecules, surface-active species, extracts and particulates, all distributed inhomogeneously throughout the bulk. In order to create the functionality expected in a given cosmetic product, several processes must be addressed and controlled; these include: the deposition of ingredients from the bulk to a surface, the timely and targeted delivery of ingredients while maintaining the appropriate physical form, the protection of an ingredient from the surrounding system until its functionality is activated, and the self-assembly or segregation of parts of a system in order to perform a specific operation. To monitor or anticipate what can happen when components are combined and as time passes is no trivial task especially when compounded with the psychophysical needs for the product to fit a specific concept and to spread, feel, pour and appear elegant.

Analytical Methods

Classical analytical methods will give the quantitative values of composition and concentration but at the expense of the spatial information. For example, in an emulsion stabilized by a lamellar structure, how the molecular species relate to each other is lost when the classical chemical analysis is performed.

Classical measurement and imaging, despite a plethora of surface sensitive techniques—e.g., electron microscopy and secondary ion emission, together with a host of different particle-surface bombardment techniques—have until recently seen a paucity of suitable noninvasive spatial methods that can examine materials in their natural, undisturbed state. 

Modern image analysis and processing, coupled with fiberoptics and imaging detectors such as slow scan charge coupled devices (CCD cameras) or spectral cameras, and combined with modern spectrometers or the scanning tunnelling devices, are in the vanguard to rectify this. This is particularly true in the UV-visible (250 to 900 µ) and near-infrared (0.7 to 2.5 µ) regions. 

Once captured in a computer-compatible form, the digitized image can be mathematically treated and significant features, such as boundaries, can be enhanced while other features of interest including fluctuations in concentration, color and shape can be extracted and quantified from the total image using various mathematical inversion techniques such as Fourier methods.¹ Coupled to this, Monte Carlo methods² can be used to feed basic physical equations for processes into a computer that then takes a stochastic pathway, i.e., a nondeterministic approach. 

The described methods and the rapid growth in molecular modelling are allowing the extraction of mechanical and dynamical properties. These advances and the growing ability to implant images of these models onto the topographical data make this field one of the future cornerstones for cosmetic scientists. 

Conventional electron microscopy (EM) has been used for many years in the cosmetics area for hard dry specimens such as pigment particles; however, aside from recent advances in electron microscopy that allow a certain amount of vapor pressure to remain above the sample, soft solids and emulsions cannot be observed in any simple way. This impasse can be partially overcome by the use of cryo-vitrification.^{3, 4} 

Cryo-vitrification eliminates many of the preparative and functional artifacts that can mislead the scientist. It involves the rapid cooling of a thin liquid layer of the sample, preventing the formation of ice crystals, which could damage the specimen. The process is noninvasive and provides clear, direct images of the unstained specimen. 

Although cryo-EM techniques are being used to collect initial data, the focus on “wet” systems has seen a re-emergence and extension of classical light microscopy in its many forms.

 The interplay between spatial resolution, time resolution, sample brightness and sample contrast is the driving force for using a particular approach and imaging technology. This has led to developments in differential interference contrast (DIC) and video enhanced microscopy (VEM),^8-11 and the various derivatives of confocal microscopy.^12-14 

DIC is an extension of classical phase contrast with the advantage to discriminate smaller differences in refractive index. It is also capable of revealing minute detail. Using VEM where the choice of camera has sensitivity (gain) and offset (zero level) characteristics, it is possible to image very small contrast changes (small grey level differences) within a specimen. The method essentially comprises stretching the image contrast by maneuvering the dynamic range to that of the differences under examination. As a consequence of this capability, not only can weakly contrasting features be seen clearly on a video screen, but very small objects also can be imaged. Traditional techniques have a resolution limit of about 0.2 µ and video-enhanced techniques have been used to detect the presence of features down to 0.04 µ, yielding ideal techniques for revealing sub-micron particles within a matrix. 

Conventional microscopy, optical or electron-based, permits excellent visualization of structures and interfacial behavior; however, the trend toward chemical distribution and state mapping has been driven by the recent availability of IR, UV, Raman and fluorescent microscopy. Together, these allow detailed viewing of a specific chemical species either by natural contrast or by some labeling mechanism. These methods are the molecular analog of the classical energy dispersive X-rays (EDX) methods for studying elemental mapping in EM. The object is viewed not in normal white light but in one of the spectral characteristics or “colors” (absorptive or emissive) of the material. 

The resultant image displayed thus is only the spatial distribution of that color alone. By changing the color in the object and repeating the process, one can map the variations in the sample^15–23 and use modern mathematical techniques to analyze the data.^{1, 24–26} Although IR microscopy preserves much of the information content of the IR experiment, current optics confine the resolution to the multimicron range. Raman microimaging, with its increased spatial resolution, preserves much more of the spectroscopic information and is potentially more attractive than IR. It has been successfully applied to the spatial composition in medium-sized emulsions, although data acquisition times make it difficult to study rapidly changing systems. The information can be collected point by point or with the correct optics and detectors imaged in one shot. 

By using time-gated luminescence,¹⁵ background fluorescence can be completely suppressed, yielding the macro image of the deposited material against a dark background. Such a capability gives a great potential for studying a low concentration of macromolecules on a variety of relevant surfaces.

The described techniques are contributing strongly to consumer product understanding, but they do not go far enough on their own. There are still problems created by scattered light, especially when attempting to recover a weak signal from within a complex three-dimensional structure. In this case, any light coming from “out of focus” sources contributes to the overall level of “noise,” reducing the contrast and ultimately wiping out the image. 

One of the most effective techniques for minimizing this problem is confocal laser scanning microscopy (CLSM),^12–14 which is commonly used for in vivo and in vitro studies.^6,7 In CLSM, a point source and a point detector, positioned in “confocality,” are used to illuminate a specific small volume of the sample and to reject light coming from any other part of the system. This means that the light coming from the focal point will be focused on and passed through the detector pinhole and registered. Light from any other part of the system will follow a different path through the optics and will not be focused on the pinhole, giving a high degree of selectivity for light emanating from the particular point. 

If the microscope is provided with a beam-scanning unit, it is possible to build up, point-by-point, a two-dimensional image in the focal plane of the microscope. This allows researchers to obtain high contrast images from regions inside thick samples. In conjunction with the strong discrimination against light from outside the focal plane, a narrow depth of focus is produced, nominally around 0.5 nm. This could be considered as providing the capability for noninvasive optical sectioning of the sample, in contrast to the physical sectioning carried out with a microtome when coupled to TEM or SEM. 

If a series of these sections are taken at different depths, it becomes possible to reconstruct, within a computer, the three-dimensional structural representation of an intact thick sample. This method has become standard fare in many laboratories and is a growing capability in many cosmetic companies.^12–14 

The lens-based methods are reaching to the very limits imposed by their fundamental design. For better resolution, a new generation of “lensless” microscopes has been developed. The methods are currently useable only in vitro but present great potential for cosmetics. The methods are known collectively as scanning probe microscopies (SPM’s)^27–33 and constitute a family of techniques that are capable of imaging to atomic resolution using a range of different molecular properties. The common feature is that the probe is mounted on a robust, piezo-electric controlled framework, which may be driven in three orthogonal directions for tens of micrometers with better than Angstrom precision.

The point-to-point data is combined in a computer to create an image of the property measured by the probe. Although high resolution work can be of value, the cosmetic chemist can work with relatively low magnifications; for example, the thickness of films on surfaces, the coverage level of coating materials, the degree of swelling of a particle, or the physical behavior of a functioning cell under the appropriate media.

Although the tip to the sample applies extremely small forces, it is possible to cause damage to soft surfaces such as proteins and surfactant layers. This can be overcome by a recent development known as non-contact, or attractive-mode atomic force microscopy (AFM). In this case, the tip-to-sample distance is increased to about 50–100 Å, where rather than the Born repulsion experienced between the tip and the sample at short spacing, the tip experiences an attractive Van de Waals force. This force is much weaker than that experienced in the repulsive-mode and the damage correspondingly diminished.

These techniques continue to revolutionize the view of surface chemistry at a small dimensional scale. Such methods will have major impact in film-forming measurements and coating processes. The development rate of different properties that can be “scanned” has been astonishing; the only limitation appears to be the ingenuity of the practitioners. The methods have been transferred to the optical region in the creation of the near field instruments (SNOM).^27–29 Although many of the more “classical” SPM methods are growing in popularity in the larger consumer companies, the optical approach still has a very long way to progress before it becomes of equal utility. It is just a matter of time.

Conclusion

The above tools do not remotely constitute a comprehensive list of what could be available in the cosmetic industry’s physio-chemical toolbox. Nothing has been discussed about bulk and surface rheology, advances in color science, or the relationship between human perception and instrumental measures. This is not to say such disciplines are not of equal worth to the cosmetic chemist, but indeed are so important that they could not be given due diligence in such a short discussion. 

Whatever happens in the next few years, it is clear that with a more discerning public and increasing government regulations, the use of modern optical tools to collect more definitive information on a product will play an increasing role in establishing which companies prosper and which falter.

 

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