Routine Absorbance Spectroscopy at the Air/Water Interface

Routine Absorbance Spectroscopy at the Air/Water Interface

JEFFREY L. MOORE, BETH A. De BRY, and KENNETH D. HUGHES* Department of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332-0400

A Langmuir-Blodgett instrument has been assembled that is capable of providing side-by-side comparisons of two separate monolayers in a true double-beam absorption geometry with no moving parts. Absorbance spectra of two different films are monitored at the air/water interface, with the use of fiber optics in conjunction with a charge-coupled-device detector and a single-stage spectrograph. The utility of this instrument is demonstrated by simultaneous measurement of monomer, dimer, and aggregate components in a single-component carbocyanine dye monolayer and a mixed carbocyanine dye/phospholipid monolayer, as the isotherm of each of these films is generated. A linear relationship between number density and the carbocyanine monomer/dimer ratio is observed.

Index Headings: Monolayer; Absorption; Carbocyanine.

INTRODUCTION

Langmuir-Blodgett (LB) techniques are frequently used for fabrication and deposition of monomolecular films. The simplicity of the technique provides a means for fabricating various monolayer and multilayer assemblies consisting of single and multiple components. These molecular assemblies have numerous applications including nonlinear optical devices and biomemitic models, as well as frequently serving as the "active" area in a wide variety of chemical sensors. These applications are reviewed in recent texts by Ulman ~ and Roberts, 2 and although they are very diverse, each requires a detailed understanding of molecular level packing geometries for optimum function.

Many proposed LB applications are based upon an optical response, and thus involve amphiphiles with ex- tended chromophore systems. Film parameters such as chromophore density and molecular orientation frequently control the photophysical characteristics of the resulting system, and therefore require detailed investigation and monitoring.

Langmuir-Blodgett monolayers containing optically active components are commonly investigated in situ by surface pressure-area measurements only. As a result, limited information is provided for the characterization of molecular orientation and two-dimensional packing geometries. For this reason, the methods of electronic and vibrational spectroscopy, in tandem with the pressure-area isotherm, have been applied to the analysis of monolayers at the interface. Spectroscopic investiga- t ions-transmission, absorption, reflection, and scatter- ing of monolayers--have the added benefit that all phys- ical disruption of the interface is avoided. 3-9

The obvious need for routine optical spectroscopy at the air/water interface has been discussed by many researchers. In 1988 Mitchell and Dluhy performed single

Received 14 September 1994; accepted 13 December 1994. * Author to whom correspondence should be sent.

reflectance FT-IR experiments at the air/water interface using a custom-built trough and a modified spectrome- ter. ~° In 1989 Duschl et al. 4 reported fluorescence microscopy experiments with a trough custom built for the microscope. Second-harmonic generation as well as UV/ Vis, IR, FT-IR, and X-ray spectroscopic methods have all been applied to the analysis of thin films. ~-14 These systems usually consist of two stand-alone instruments with little if any integrated functions. In the case of vacuum techniques, analysis is performed on deposited films. Although these techniques have been successfully imple- mented by the individual researchers, progress has been slow in the development of commercial systems that fa- cilitate routine spectroscopic analysis.

Combining LB monolayer construction and optical spectroscopic analysis involves a variety of difficulties. In many cases, several hours may be required for an isotherm to be generated under equilibrium conditions, and thus source drift is commonly a problem with scan- ning systems? Utilization of multiple detectors, in com- bination with fiber optics or mirrors, does allow double- beam absorption spectroscopy to be performed during isotherm generation, but this method certainly cannot be considered routine. Use of a diode array and a chopper has recently been demonstrated as an acceptable alter- native to the latter system. 9

A Langmuir-Blodgett instrument with integrated spectroscopic capabilities has been assembled. The instrument provides unattended spectroscopic examination of monomolecular films and supra-molecular assemblies at the air/water interface. Specifically, this instrument is capable of providing side-by-side comparisons of two separate monolayers in a true double-beam geometry. In single-beam geometries, the instrument is capable of monitoring absorption or emission of two different monolayers at the air/water interface as well as the monolayer(s) transferred to a solid substrate. The instrument is com- prised of a commercial Langmuir-Blodgett trough inter- faced with a charge-coupled device (CCD) detector, a single spectrograph, fiber optics, and a xenon arc lamp. All components of the instrument are controlled from a single computer station using a graphical interface pro- gramming language.

Simultaneous analysis of 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) as a single component monolayer and in a mixed monolayer with DL-a-dipamitoyl-phosphatidylcholine (DPPC) is reported as an initial dem- onstration of this instrument. Carbocyanine dyes are cur- rently used as biological probes for membrane potential. ~5 Probe function is based upon changes in the aggregation characteristics of the dyes in the membrane. Investigation of carbocyanine and derivatives in LB monolayers provides an avenue for understanding current membrane "staining" experiments and will aid in the development of improved membrane probes.

886 Volume 49, Number 3, 1995 0003-7028/95/4903-038652.00/0 APPLIED SPECTROSCOPY © 1995 Society for Applied Spectroscopy

~ t ll llll l l l l l l l l l l l Ilill

II Illl Ill l l l l Ill I l i l l l

(A)

er Optic

. M

~ D e t e c t i o n . / / [ Fiber Optic

M

(B) FIG. 1. (A) Block diagram of LB trough, and aluminum reference trough. P, pressure sensors; D, dipper mechanism; M, mirror; B, barrier; S1, compartment A, and $2, compartment B. (B) Fiber optic mount. Source, detector, and fiber-optic arrangements are described in the text.

EXPERIMENTAL

Figure 1 provides a partial schematic diagram of the instrument, which is composed of a Langmuir-Blodgett trough, a xenon arc lamp, an imaging spectrograph coupled to a CCD detector, and a single Macintosh IIci that controls all components of the instrument.

Langmuir-Blodgett Trough. A circular LB trough purchased from Nima Technology Limited (Model 2022, Fromherz type) was used for all investigations. The trough is milled from a solid piece of PTFE and is mounted on a stainless steel base, and it incorporates an inflated air cushion to minimize vibrations. This alternate-layer trough consists of two film compartments and a dipping compartment. The three-inch well and dipping mechanism completely separates the two main compartments. Two independently addressable stepper-motor-driven PTFE barriers are used for film compression. Pressure sensors for each film compartment utilize the Wilhelmy plate method of surface tension measurement. The trough is housed in a laminar flow hood installed with HEPA filters (Laminaire Corporation).

Optical Components. A xenon arc lamp (Oriel Corp.) was used as the excitation source. Neutral-density filters (Melles Griot) were used in addition to a computer-controlled filter wheel (Oriel Corp.) to appropriately condi- tion the excitation range (200-850 nm) and intensity. A single-stage imaging spectrograph fitted with a fiber-optic adaptor (Jobin-Yvon Optical Systems, Model CP200) was used. This spectrograph incorporates a holographic con- cave grating, with a groove density of 200 lines/ram and a spectral range of 200-850 nm. A liquid nitrogen-cooled

A Sample 1

B Reference

C Sample 2

Wavelength FIG. 2. CCD spatial assignments. Heavy black areas indicate nonil- luminated areas of the chip.

CCD detector with an EEV 298 x 1152 chip (LN/CCD- 1152, Princeton Instruments) was used for detection.

Optical Arrangement. Aluminum mounts, designed and machined in-house, were used to suspend one leg of an excitation and corresponding collection fiber optic above each film compartment of the trough. Trifurcated metal cable fiber optics (random fiber orientation) were used for excitation with lenses (75-mm focal length, Oriel Corp.) mounted on the end of both excitation and collection fiber optics. Lenses were held at 30 ° with respect to the normal. The collection fiber optic (Princeton Instru- ments) was oriented in a linear array at the spectrograph slit. Top, middle, and bottom fibers of this array corre- sponded to each leg of the fiber opti c . Each leg of the collection fiber corresponds to a unique area of the CCD chip (Fig. 2). These components allow the creation of "three" separate detectors on a single CCD chip. Mirrors were placed between the excitation and collection lenses on the underside of each suspended mount (Fig. 1), and a corresponding mirror was placed in the trough subphase. By varying the height of each mount it is possible to increase the number of reflections between optics, thus increasing the film/radiation interaction. The absorption spectra in Figs. 3 and 4 were collected with the use of four reflections or eight interactions with the film/water interface. A small aluminum reference trough situated next to the main trough utilized the third leg of the trifurcated fiber optics.

Computer Control. A Macintosh IIci (Apple Comput- ers) running LabVIEW (National Instruments), was used for control of all LB trough and spectroscopy operations. The trough is controlled through an RS-232 interface and the detector is controlled through a GPIB interface.

Chemicals. 3,Y-Dioctadecyloxacarbocyanine perchlorate was purchased from Molecular Probes Inc. DL-o~- dipamitoyl-phosphatidylcholine was purchased from Sig- ma and stored at 0°C (see Fig. 5). Reagent-grade chloroform (Fisher Scientific) was distilled directly before use in a greaseless distillation apparatus. Bamstead Nanopure water (t8.3 MrS resistivity) was used for the trough subphase. All manipulation tools, including tweezers, gas- tight microliter syringes (Hamilton), and glassware used in the vacuum apparatus, were rinsed with distilled chloroform several times prior to use. Powder-free Class 100

APPLIED SPECTROSCOPY 387

0 0

30-

zo- r ~ r ~

lO-

0 o io 16o 30 60 ~J0 120 150 180

Area/molecule (Angstroms) z

(A)

0.04507

0.04001

0"03501 o 0.0300-

e~ 0.0250-

0.0200-

< 0.0150-

0.0100-

0.0050-

0.0000-

-0.0050-

-0.0100- 380.0 400.0 420.0 440.0 460.0 480.0 500.0 520.0 540.0 560.0 580.11

Wavelength (nm) (B)

FIG. 3. (A) Pressure-area isotherm of a single-component DiO monolayer. (B) Multiplot of absorbance spectra obtained during isotherm generation. Pressures: A = 0.0 raN/m; B = 3.0 raN/m; C = 9.0 raN/ m; D = 15.0 raN/m; E = 21.0 raN/m; and F = 27.0 mN/m.

30

,-, 25-

g 15-

10-

5-

I

,~0 60 80 100 120 140 Area/molecule (Angstroms) 2

40-

(A) 0.0400]

0.0350J

0.03001

0.0200-t

~ 0.0150.

~ 0.0100-

0.0050.

0.0000.

-0.0050.

-0.0100- 380.0 40b.0 42b.0 44b.0 460.0 48~1.0 50b.0 520.0 54b.0 56b.0 51 '.0

Wavelength (nm)

(B) FIo. 4. (A) Pressure-area isotherm of a mixed dye/phospholipid monolayer. (B) Multiplot of absorbance spectra obtained during isotherm generation. Pressures: A = 0.0 naN/m; B = 3.0 raN/m; C = 6.4 raN/m; D = 14.00 raN/m; E = 18.9 raN/m; F = 22.7 mN/m.

cleanroom gloves (Oak Technical) were worn at all times. Chloroform solutions of DiO and DPPC were manually spread dropwise on the trough for monolayer formation.

Isotherms. Isotherms of DiO and DPPC were generated under the following conditions: For dual-compartment studies, both a pure dye solution and an approximately 1:1 DiO/DPPC solution were prepared. The solvent for all solutions was distilled chloroform. The pure DiO solution was prepared by dissolving 3.21 mg of DiO (mol wt 882 g/mol) in 10.00 mL of distilled CHC13 for 0.321- mg/mL concentration; 17.16 mg of DPPC (734 g/tool) was dissolved in 50.00 mL of distilled CHC13 for a 0.3432- mg/mL solution; and 5.00 mL of the 0.321 DiO solution was mixed with 5.00 mL of the 0.3442-mg/mL DPPC solution. The DiO/DPPC mixture was 46% DiO molecules and 54% DPPC molecules with an average concentration of 0.3476 mg/mL and an average molecular weight of 802 g/tool. Dipamitoyl-phosphatidylcholine has been well characterized since monolayers and bilayers of lipid films provide good model systems for biological membranes. Isotherms generated in this work compare well with literature results. 1°-12,14

Absorbance Spectroscopy. Simultaneous spectroscopic analysis was performed on both the single-component

CH3 CH 3

(A)

O

(CH3)3 NCH2CH20 P

C_O_H~2(CH2) 14CH3

O CH2CH CO2(CH2) 14CH 3

(B) FIG. 5. Molecular structures of (A) 3,Y-dioctadecyloxacarbocyanine perchlorate (DiO) and (B) dipamitoyl-phosphatidylcholine (DPPC).

388 Volume 49, Number 3, 1995

DiO film and the DiO/DPPC mixed film during monolayer compression. A double-beam absorption geometry was used in all cases. A blank spectrum was collected prior to the spreading of the solutions, which was used to calculate a balance spectrum for each film compartment. The balance spectrum is used to adjust for different response curves for each window on the CCD chip as well as differences in pathlength. The balance spectrum is a ratio of the response of the reference window and each sample window. After the blank was collected, the single- component DiO solution was spread on one compartment (barrier completely open) and the DiO/DPPC mixture was spread on the other compartment (barrier completely open). The solvent was allowed 30 rain to evaporate. Both monolayers were compressed at a speed of 10 cmZ/min, and absorbance spectra were collected at 3-mN/m pressure intervals. Integration time for each spectrum was 1.5 s, and five exposures, at each pressure interval, were av- eraged.

RESULTS AND DISCUSSION

Analysis of monolayer films usually involves monitoring the surface tension of the subphase, which decreases with a reduction in the mean molecular area of the film constituents. Correlating the pressure of the film to the space occupied by each individual molecule at the surface (known as the area/molecule) results in a plot known as the pressure-area isotherm. 1.2 Unfortunately pressure-area isotherms do not provide sufficient information for a detailed molecular level interpretation of film behavior. Since many "active components" in monolayer and multilayer devices and model systems are amphiphilies containing chromophores, the use of absorption and emission techniques, in tandem with the isotherm, is advantageous. Analysis of the isotherms in Figs. 3 and 4 does little to reveal the complexity and variety of molecular interactions that are obviously shown to be present by investigation of the absorption spectrum.

The hybrid LB/spectroscopy system partially illustrat- ed in Fig. 1, and described in the Experimental section, utilizes software to integrate two very different instruments. The LB trough is controlled via an RS-232 serial interface which must be continuously addressed for con- stant pressure control. As a result, trough commands must be sent repetitively to maintain control of barrier speeds and positions. In addition, continuous recording of the isotherm requires a repetitive measurement of barrier position and surface pressure.

When one is sending commands or transferring data from the CCD detector, an interruption in communication with the trough, for a period of time longer than approximately three seconds, results in a "timing out" of communication with the trough. This "timing out" results in a loss of pressure control and stoppage in barrier movement, until communication is restored, which can distort the isotherm. To avoid these problems, it was necessary to design the software to limit the time the detector is being addressed.

The spectroscopy component of the system has only a single addressable component, the detector, which is controlled via a GPIB. Unlike the trough, the detector commands may be sent once and stored in a controller RAM.

Therefore, each set of detector commands is interlaced with commands sent to the trough. The ability to operate the detector in this "latched" mode improves the quality of the isotherm and the overall performance of the hybridized LB/spectroscopy system.

In operation, this hybridized system is fully automated. A repetitive software loop that receives and processes data as well as sends updated compression parameters is used for LB trough control. Absorbance spectra are obtained as the isotherm is generated by keying on a specified pressure interval. Each time the pressure increases by the specified interval, a trigger is sent to the detector to begin exposures. While the detector is integrating, the trough is readdressed and compression of the monolayer continues. The software now monitors the GPIB for a service request. When the system detects this request, a transfer command is generated and the data stored in the detector RAM are transferred. After data transfer, the trough is re-addressed, and spectral manipulation (av- eraging, absorbance calculation) is then initiated in a step- wise, interlaced fashion. Data manipulation requires a significant amount of time and does result in a loss of pressure control and isotherm distortion if the tasks are not executed in this manner. Compression rates between 10 and 20 cm2/min, which correspond to barrier rates of 2-4 mm/min, are easily obtained. With respect to the user interface, the isotherm is continuously displayed and updated on the screen, while the spectra are continuously displayed but only updated after a new spectrum has been acquired.

Although the software provides an apparently seamless hybridization of instruments, the other critical system component is the imaging spectrograph, which makes it possible to fully utilize the two-dimensional nature of the CCD array. Each arm of the trifurcated fiber optic is imaged on a spatially distinct area of the CCD chip, in effect giving three unique detectors with a central control (Fig. 2). This optical arrangement allows for true simultaneous double-beam spectrometry of two different films at the air/water interface (with the third leg serving as a reference). A comparative analysis between a sample and reference monolayer, such as a film with an active component doped into a host matrix, with the host matrix as a reference on the other compartment, may also be performed. For stable excitation sources, single-beam spectroscopy can be performed on the two monolayers at the air/water interface and the transferred monolayer(s). All configurations are available by proper assignment of the fiber-optic legs.

Instrument capability has been demonstrated by simultaneously monitoring the isotherm and absorbance spectra at several positions on the isotherm for a single- component and mixed monolayer of a carbocyanine dye and dipamitoyl-phosphatidylcholine, simultaneously. A double-beam geometry is used to collect both absorbance spectra. Carbocyanine dyes are strong absorbers (e = 2 x 106) 16 and have current application as fluorescent biological probes. 3,3'-Dioctadecyloxacarbocyanine perchlorate readily forms monolayers at the air/water interface and has been characterized at the air/water and air/substrate interfaces. 7,17-2° In both of these environments the photophysical characteristics of systems incorporating this and related molecules are complex. Monomers, dimers,


and aggregates in varying ratios depending on fabrication conditions are all possible and are usually present. Car- bocyanine derivatives of DiO with shorter alkyl chains are used as cationic membrane probes for potential gra- dients. These molecules accumulate in cell membranes, where they undergo absorption and emission changes due to dimerization and aggregation resulting from the membrane potential. Derivatives have also been used to vi- sualize the endoplasmic reticulum in both living and fixed cells, have been used as low-molecular-weight tracers to define neuronal connectivity, and have been used to fol- low liposome fusion and cell division. These dyes have been observed to stain an entire membrane via lateral diffusion when the dye is applied locally. A summary of this literature is available in Ref. 16. The ability to ma- nipulate the photophysical as well as the diffusional characteristics of these probes is important for current membrane investigations and the development & n e w probes.

In our work, a single-component DiO solution was spread on one compartment while a solution of the DiO/ DPPC mixture was spread on the other compartment of the LB trough. The monolayers were compressed at a speed of 10 cm2/min, and absorption spectra were collected at 3-mN/m intervals along the isotherms. Total time for analysis was approximately 2.5 h. Analysis of these two different films separately on a single layer trough would require a correspondingly longer time.

Comparison of the pure DiO (Fig. 3) and DiO/DPPC mixture (Fig. 4) absorbance spectra indicates differences as well as similarities in the packing of the molecules in each monolayer. These molecular complexities are not apparent from the isotherms alone. The peak at 500 nm is due to monomer absorption, while the peak at 469 nm results from dimer absorption. 1' Carbocyanine dyes can form J-aggregates, indicated by a red or bathochromic shift of the monomer, and H-aggregates, indicated by a blue or hypsoehromic shift of the dimer. Packing models for these aggregates have been described previously. 2' Formation of these aggregates requires that the long axes of the chromophores overlap; therefore the long axes must lie in the same plane, parallel to each other. Orientation studies indicate that the long axis of the ehromophore lies in the plane of the interface, while the short axis rotates from in the plane to normal to the plane as the pressure increases./z,23 As the pressure or number density of molecules increases, and the short axis tilts to the normal, dimer and aggregate formation should increase as the chromophore groups pack closer.

In the single-component DiO monolayer (Fig. 3), the monomer shows an approximately 5-rim red shift, indicating J-aggregation, while the dimer displays an approximately 16-nm blue shift and broadening, indicating the presence of H-aggregation. As the molecular number density increases during compression, the dimer absorption begins to exceed that of the monomer peak. The spectra at higher pressures indicate that there are several species of dye in the monolayer, including monomer, dimer, and both types of aggregates. As the molecular number density increases, a greater number of molecules are present as dimers and aggregates, and thus a decrease in the monomer concentration is observed.

In the mixed DiO/DPPC monolayer (Fig. 4), there is little shift in peak maxima for monomer and dimer, thus

1.6

1.4

,~ 1.2

.E 1

0.8

0.6

0.4 1.50E+14 2 . 2 5 E + 1 4 3 .00i~+14 3 . 7 5 E + 1 4 4.50E+14

# molecules /cm 2

FIG. 6. Monomer/dimer absorbance ratio as a function of molecular number density for the single component monolayer. Values plotted are single wavelength points obtained from the spectra in Fig. 4.

indicating less aggregation in comparison to that for the single-component DiO layer. The dimer peak absorption still increases with respect to the monomer, but the increase is smaller than that in the single-component DiO monolayer. This observation is another indication that aggregation has decreased in the mixture. However, if dye molecules and lipid molecules were soluble in one another, we would expect a "checkerboard" or alternating molecule arrangement at the interface and a significantly reduced concentration of the dimer component. Since this is not the case, phase separation of the two components is suggested. Observation of these separated phases or "islands", in similar systems, has been conducted with optical microscopy and with atomic force microscopy (AFM) fl 4

It is also expected that a dilution of the dye by 50% in the mixed monolayer would decrease the absorbance intensity by a corresponding amount. Comparison of the absorbance intensities of monomer peaks in the single- component monolayer and the mixed monolayer does not indicate the expected scaling of intensity, and again provides evidence that phase separation in the mixed layer is occurring. Comparison of the area under the absorption curves for all forms of the dye present, in the single component monolayer and the mixed monolayer, does indicate a reduction of the total dye component in the mixed monolayer. It is quite possible that a simple analysis of this type could be used to rapidly characterize phase separation in these films. More detailed investigations of this type are ongoing.

Finally, graphing the monomer/dimer absorbance ratio (Fig. 6) as function of the molecule number density shows that the monomer/dimer ratio continually decreases in a linear manner during compression. This relationship also holds for the mixed monolayer (data not shown). These results compare well with those from previous work which used single-point absorption measurements of the monomer early in the isotherm and dimer absorbance late in the isotherm. TM

The instrument described facilitates the simultaneous and rapid spectroscopic analysis of different monolayers at the air/water interface. Collection of a full UV/Vis

390 Volume 49, Number 3, 1995

absorp t ion spec t rum dur ing i so therm genera t ion is a pow- erful means o f observ ing and character iz ing the molecular-level packing geometr ies occurr ing in the monolayer . The optical geomet ry o f this i n s t rumen t can be easily modi f ied to measure two different m o n o l a y e r films in a d o u b l e - b e a m geomet ry or two m o n o l a y e r s at the a i r /water interface and the t ransferred monolayer(s) , in a single- b e a m geometry . M o n o l a y e r analysis o f the in teract ions between lipid c o m p o n e n t s and m e m b r a n e probes is pro- v id ing i m p o r t a n t infi~rmation which can be appl ied to under s t and ing m e m b r a n e b e h a v i o r and probe localiza- t ion character is t ics and will lead to the design o f new m e m b r a n e probes. These analyses are t radi t ional ly a very slow and t ime- in tens ive endeavor . It is h o p e d that hybr id ins t ruments o f this type will increase the t h roughpu t o f such invest igat ions.

ACKNOWLEDGMENTS

We acknowledge Ken Williams in the Georgia Institute of Technol- ogies machine shop for aiding in the design and construction of the optical mounts. B. A. DeBry gratefully acknowledges partial support from the Georgia Tech Center for Biosciences and Biotechnology.

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Schafer, J. Sondermann, W. Sperling, P. Tillmann, and J. Wiegand, Mol. Cryst. 2, 199 (1967).

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Thin Solid Films 68, 191 (1980). 14. D. Mobius, Ber. Bunsenges. Phys. Chem. 82, 848 (1978). 15. P. J. Simms, A. S. Waggoner, C.-H. Wang, and J. F. Hoffman,

Biochemistry 13, 3315 (1974). 16. R. P. Haugland, Molecular Probes Handbook of Fluorescent Probes

and Research Chemicals (Molecular Probes, Eugene, Oregon, 1992), 5tb ed.

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Documents

Routine Absorbance Spectroscopy at the Air/Water Interface