1 Moscow State University, RussiaFaculty of physics and International Laser Center RAMAN AND ATR FTIR SPECTROSCOPY IN THE STUDY OF CHEMICAL REACTION RATES I.A. Balakhnina, N.N. Brandt, A.Yu. Chikishev, A.A. Mankova, I.G. Shpachenko

2 How to measure reaction rate?Reaction rate = Rate of changes in reagent (product) concentration Optical methods Disadvantages PHOTOMETRY Overlapping absorption bands POLARIMETRY Optically active substrate or product FLUORIMETRY Overlapping absorption bands and concentrational limitations Universal method is unavailable Task: Experimentally prove the applicability of vibrational spectroscopy in the measurements of chemical reaction rates

3 Reaction of alkaline hydrolysis of ethyl acetate: Raman spectroscopy of the componentsFrequency сm-1 Component 637 CH3COOC2H5 852 880 C2H5OH 929 CH3COOK 1345 1413 1453 CH3COOC2H5 , C2H5OH 1736 Raman spectrum of the reaction and its approximation with linear combination of Raman spectra of the components: ethyl acetate (CH3COOC2H5), ethanol (C2H5OH) , potassium acetate (CH3COOK), and KOH.

4 Processing of Raman data seriesIntensity kinetics Reaction rate k

5 Effect of background subtraction and scaling

6 Temperature dependence of reaction rates22°С 42°С k, М-1с-1 0,048±0,004 0,28±0,02 Raman spectra of the reaction mixture (hydrolysis of ethyl acetate), measured at s from the beginning of reaction at a temperature of 22 °C Time dependence of the integral Raman intensity of the band at 880 cm-1 measured at different temperatures An increase in temperature leads to an increase in reaction rate

7 Reaction rate and activation energyА – const Еа – activation energy R – gas constant Т – temperature Reaction rates of ethyl acetate hydrolysis at different temperatures

8 Reaction rate and activation energy

9 Reaction rates of alkaline hydrolysis of ethyl acetate determined using different spectral linesTime dependences of Raman intensity of the bands at 880, 929, and 852 сm-1 at a temperature of 25 °C and their approximations Raman spectra of the reaction of ethyl acetate hydrolysis, measured at seconds from the beginning of reaction at a temperature of 25 °C Mean value of the reaction rate is k = 0,088 ± 0,006 M-1s-1

10 Raman spectroscopy of spontaneous and enzymatic hydrolysisRaman spectra of the reaction of enzymatic hydrolysis of 2,4-dinitrophenylacetate (DNPA) in the presence of chymotrypsin, measured at 100 s min from the beginning of reaction 14°С pH 7,8 Enzymatic hydrolysis: Spontaneous hydrolysis: KM = (39,55±0,01)10-4 M kh = (1,26±0,01)10-5 s-1 S – substrate; E – enzyme; P – product; ES – enzyme-substrate complex

11 ATR FTIR geometry ATR crystal (diamond) Reflected IR lightIncident IR light Evanescent wave d

12 Reaction of alkaline hydrolysis of ethyl acetate: ATR FTIR spectroscopy of the componentsFrequency сm-1 Component 1022 CH3COOK 1050 CH3COOC2H5 C2H5OH 1265 1380 EtOAc 1414 1553 ATR FTIR spectrum of the reaction mixture at 17 s and the spectra of the components: ethyl acetate (CH3COOC2H5), ethanol (C2H5OH) , potassium acetate (CH3COOK), and KOH

13 Reaction rates: ATR FTIR spectroscopyATR FTIR spectra of the reaction of ethyl acetate hydrolysis, measured at s from the beginning of reaction at a temperature of 27,4 °C Time dependence of the area under the band at 1265cm-1 measured at 27.4 °C and its approximation ATR FTIR T, °C 25,6 27,4 27,8 k103, М-1с-1 490±10 540±10 830±10 480±10 Raman 25 89±8 94±8

14 ATR FTIR time-dependence: liquid-in-liquid solutionConcentration changes are obvious Solvent evaporation? Interaction with ATR crystal surface? Molar ratio: Ethanol / Benzoic ether = 5

15 In case of reaction: product increasing rate = reagent decreasing rateATR FTIR time-dependence: solid-in-liquid solution Molar ratio: Acetonitrile / DNPA = 100 In case of reaction: product increasing rate = reagent decreasing rate

16 Reaction rates: ATR FTIR spectroscopy

17 Reaction rates: ATR FTIR spectroscopy1264, 1300, 1359, 1393, cm-1 unchanged 607, 634, 785, 847, 916, 938, 1097, 1115, 1373 cm-1 – intensity 1045, 1238 cm-1 (skeletal) – intensity and width Ethyl acetate ordering (crystallization) on diamond surface

18 Conclusions Raman spectroscopy makes it possible to locally measure the rates of chemical processes (including enzymatic) in experiments with liquids and solids in the absence of specific requirements to reagents (substrates) Raman measurements make it possible to determine rate constants using variations in intensities of several spectral bands, so that the accuracy of calculations can be increased In the case of ATR FTIR measurements, the phase transition of liquids into quasi-crystalline state on the surface of ATR crystal is possible (even for diamond)

19 Взаимодействие гомогенных растворов с алмазомИК спектры 2,1 М этилового эфира бензойной кислоты (этилбензоата) в этиловом спирте из серии, измеренные через 15(черная кривая), 100 (красная кривая) и 200 секунд (синяя кривая) после начала измерений на кристалле НПВО. ИК спектры 190 мМ раствора 2,4-динитрофенил ацетата в ацетонитриле из серии, измеренные через 3(черная кривая), 100 (красная кривая) и 200 секунд (синяя кривая) после начала измерений на кристалле НПВО. ИК спектры фосфатного буфера из серии, измеренные через 3 (черная кривая), 100 (красная кривая) и 200 секунд (синяя кривая) после начала измерений на кристалле НПВО. Все спектры совпадают.

20 ATR FTIR spectroscopy of spontaneous and enzymatic hydrolysisFour reaction kinetics at 1380 см-1 ATR FTIR spectra of spontaneous reaction of 100 mM ethyl acetate hydrolysis, measured at s from the beginning of reaction at the temperature of 23,5 °C ATR FTIR spectra of ethyl acetate measured at 3, 100, and 200 s after the beginning of reaction