4B08

pH at the Lipid/Water Interfaces Revealed by Heterodyne-DetectedElectronic Sum Frequency Generation(Mol. Spectrosc. Lab., RIKEN) KUNDU, Achintya; YAMAGUCHI, Shoichi; TAHARA, Tahei
The biological cell membrane is a complex mixture of lipids, proteins, carbohydrates, andcholesterols. The pH and polarity at the cell membrane are very important for many biologicalprocesses, such as proton transfer, ion transfer, etc. Despite their importance, however there is no
systematic study of the pH and polarity at the cell membrane. Because the complexity of the real cellmembrane makes it difficult to probe, we use a lipid monolayer on a water surface (air/lipid/waterinterface) as a model of a cell membrane. We report that heterodyne-detected electronic sum
frequency generation (HD-ESFG) with a surface-active pH indicator dye allows us to estimate thepH and polarity at the model cell membrane.

aC17H35

C17H35HOO

O-OO

The most reliable method of determiningH++bulk pH is UV-visible spectrometry with a pHOBase (A-)

Acid (HA)

indicator dye. It is also possible to apply
spectrometry to the lipid/water interface by usingb

HD-ESFG, which can provide the second-ordernonlinear optical susceptibility (χ(2)) of theinterfacial molecules. Using this technique, wesystematically determine the pH at lipid/waterinterfaces. Figure 1a shows the equilibrium theOOOOOOOHOHOO- P OO

HODPG

OOO
H

heptadecyl-7-hydroxycoumarin (HHC). StudiesO-O
O P OOOH

N+DPPC

acid (HA) and base (A–) of a pH indicator, 4-OOO

four model lipids, shown in Figure 1b: 1,2-HN+OH

DPPG

were performed at the water surface with each ofODPTAP

Figure 1. a, Acid-base equilibrium of thesurface-active pH indicator, HHC. b, Chemicalstructure of the four model lipids: DPG(nonionic), DPPC (zwitterionic), DPPG
(anionic) and DPTAP (cationic).

dipalmitoyl-sn-glycerol (DPG, nonionic), 1,2dipalmitoyl-sn-glycero-3-phosphocholine (DPPC,zwitterionic),

1,2-dipalmitoyl-sn-glycero-3-phospho -(1'-rac-glycerol) (DPPG, anionic), 1,2dipalmitoyl-3-(trimethyl-ammonium)(DPTAP, cationic).

propaneFigures 2a and 2b show the surface-selective Im[χ(2)]and Re[χ(2)] spectra of the indicator at the DPG/waterinterface, with the bulk pH regulated by sodiumhydroxide. By analyzing the bulk-pH dependence of theχ(2) spectra, the bulk pH at which [HA] = [A–] isdetermined as 9.6. Generally, the pH is equal to the pKa(negative logarithm of the acid dissociation constant)when [HA] = [A-]. However, this bulk pH at which [HA]= [A-] is not the pKa of the indicator at the DPG/waterinterface, because the pH is measured in the bulk. If weanalyze the χ(2) spectra against the interfacial pH, we canobtain the pKa. Assuming interfacial pH = bulk pH + ∆,pKa is given as 9.6 + ∆.
The pKa is equal to the standard reaction Gibbs
energy of HA→ H+ + A– divided by RTln10. The
standard reaction Gibbs energy is given as a function of
theeffective

relative

dielectric

constantof

thesurrounding medium which can be estimated by theFigure 2. a, b, Surface-selective
electronic spectra of the pH indicator atthenonioniclipid(DPG)/waterinterface. The imaginary and real partsof χ(2) at different pH are shown in a andb, respectively.

solvatochromism of A–. The peak wavelength of the Im[χ(2)] spectrum of A– in Figure 2a allows us
to estimate the effective relative dielectric constant of the DPG/water interface as 12. From thiseffective relative dielectric constant, the pKa of HHC at the DPG/water interface is evaluated as 11.6.
Because this pKa should be equal to 9.6 + ∆, ∆ is obtained as +2.0. In the same manner, the values of∆ for the other lipid/water interfaces are obtained as shown in Table 1. This work clearly shows thatthe interfacial pH is substantially deviated from the bulk pH and that is changes largely depending on
the head group of the lipids.
Table 1. Peak position of the base (A–), effective relative dielectric constant, and pH difference .
Interfacepeak nmEffective relative
dielectric constantInterfacial pH - bulk pH

DPG/water

377

12+2.0DPPC/water375

22-1.1

DPPG/water365

41-2.6DPTAP/water376

20+4.4