888 Volume 55, Number 7, 2001 APPLIED SPECTROSCOPY 0003-7028 / 01 / 5507-0888$2.00 / 0 q 2001 Society for Applied Spectroscopy Study of Thermal Melting Behavior of Microbial Polyhydroxyalkanoate Using Two-Dimensional Fourier Transform Infrared Correlation Spectroscopy GE TIAN, QIONG WU, SUQIN SUN, ISAO NODA, and GUO-QIANG CHEN* Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China (G.T., Q.W., G.-Q.C.); Department of Chemistry, Tsinghua University, Beijing 100084, China (S.S.); and The Procter and Gamble Company, 8256 Union Centre Blvd., West Chester, Ohio 45069, USA (I.N.) The molecular level premelting process of puried biosynthesized polyester poly(hydroxybutyrate-co-hydroxyvalerate) or P(HB-co- HV) (20.4% HV) was investigated by two-dimensional Fourier transform infrared (2D FT-IR) correlation spectroscopy. Intensity variations and band shifts in the characteristic spectral regions for C5O groups (1710–1770 cm 21 ), C–H groups (2910–3010 cm 21 ), and C–O–C groups (1220–1310 cm 21 ) were selected for a detailed study of the thermally induced phase transition of the copolymer. The 2D correlation approach successfully demonstrated that a fully amor- phous liquid-like structure was formed following the disappearance of the crystals in the sample, by going through an intermediate state related to the lost conformation of the highly ordered and helical polyester main chain. Index Headings: Polyhydroxyalkanoate; PHA; Poly(hydroxybuty- rate-co-hydroxyvalerate); P(HB-co-HV); 2D FT-IR; Premelting; Biodegradable polymer. INTRODUCTION Microbial polyhydroxyalkanoates, abbreviated as PHAs, are aliphatic semicrystalline polyesters made by various microorganisms. They are believed to have many potential industrial applications such as biodegradable materials, tissue engineering, controlled release devices, etc. 1–4 The structure of PHA is illustrated in Fig. 1A and 1B. Fourier transform infrared (FT-IR) spectroscopy was applied as a novel noninvasive analytical probe to qual- itatively study PHA. 5 PHA shows a strong FT-IR band near 1730 cm 21 attributed to the C5O stretching vibra- tion, and accompanying bands near 1280 and 1165 cm 21 due to the C–O–C group. The crystallinity and the melt- ing behavior of PHA are especially interesting for many researchers as they are related to many potential appli- cations of this polymer. 1,2,6,7 In this report we used two- dimensional infrared (2D IR) spectroscopy to elucidate the detailed thermally induced melting process of PHA samples. The basic concept of two-dimensional infrared spec- troscopy was rst introduced by Noda in 1986. 8 In this approach, the synchronous and asynchronous 2D IR spectra plotted as a function of two independent wave- number axes are constructed from the perturbation-in- duced time-dependent uctuations of IR signals. The ap- pearance of correlation peaks in synchronous and asyn- chronous 2D spectra represents the overall similarity and dissimilarity, respectively, of the basic trends of dynamic Received 29 November 2000; accepted 9 March 2001. * Author to whom correspondence should be sent. intensity variations observed at two different wavenum- bers. 9 Some distinct advantages of the 2D correlation ap- proach over conventional one-dimensional FT-IR spec- troscopy have been identied. Correlation peaks located along the diagonal in synchronous spectra, named auto- peaks, represent the extent of the dynamic variation of the IR signal induced by the external perturbation. 10 They are used as measures of the susceptibility of the IR in- tensity variation to a given perturbation. Cross peaks in synchronous spectra are especially useful for identifying the various inter- or intramolecular interactions. This is because the simultaneous variations of IR signals at dif- ferent wavenumbers often indicate the commonality of the origin of IR signals, e.g., identical or strongly inter- acting functional groups or moieties placed in a similar molecular environment. 11,12 Asynchronous spectra are rst utilized to enhance the spectral resolution, for if the IR intensities of highly overlapped bands vary in differ- ent ways under the external perturbation, they can be clearly distinguished by the presence of asynchronous cross peaks. 9 The signs of the synchronous and asyn- chronous correlation peaks point out the sequential order of the responses of the system components to the external stimulus, which has proven to be another powerful ad- vantage of 2D analysis. 13,14 In the earlier days of 2D correlation spectroscopy, the type of perturbation used to induce the dynamic varia- tions of IR signal intensities was limited to simple sinu- soidal perturbations, like a repetitive mechanical defor- mation or electrical stimulus. A more general 2D corre- lation analysis method was proposed in 1993, which made it possible to expand the utility of 2D correlation spectroscopy by employing any arbitrary perturbation, enabling much wider ranges of applications. 15 The spec- tral intensity uctuations can be induced by many kinds of physical variables such as temperature, pressure, con- centration, and composition, as well as other external stimuli. 15 The 2D correlation approach has received in- creasing attention in recent years and has been very suc- cessful in several research elds of study, including the study of biological samples. 16–19 It is natural to extend the application of this versatile analytical technique to the study of PHA. The physical properties of PHAs have been widely studied for their potential as biodegradable and biocom- patible thermoplastics. Along with the homopolymer, po- lyhydroxybutyrate (PHB), the random copolymers of hy- droxybutyrate-co-hydroxyvalerate have been most exten-