Polarimeter Sample Results
Below are some experimental results obtained using the Propeller Polarimeter.
Warm-Up DriftHere are optical rotation measurements for a blank recorded on two different instruments as a function of time after instrument power-up.
With the instrument zeroed immediately after switching on, one observes (blue trace) an initial drift of 0.050 over the first hour of operation with readings then stabilizing.
The standard deviation in the optical rotation readings taken over an 8 hour measurement period after the initial warm-up stage here is ~ 0.010.
Quartz Calibration Plate ResultsTo check the accuracy of the polarimeter we used a Rudolph quartz calibration plate whose optical rotation is certified to an accuracy of 0.0010 at various wavelengths. This calibration plate is actually comprised of two plates - #8393 : +10.9940 and # 8114 : -21.9790 ; in combination these yield an optical rotation of -10.9850 at 589.44 nm.
The data opposite has been obtained by carefully tilt-tuning an interference filter in front of the yellow LED so as to accurately set the measurement wavelength to that of the Na "D" lines.
After, initially zero-ing the instrument, the measurement results (quoted with standard deviations) agree to within an accuracy of 0.01-0.020 in each case.
Sucrose Calibration CurvesBy measuring a sample’s optical rotation θ (at wavelength λ) at a known concentration c (g/ml) in a cell having path length l (dm) its specific rotation can be determined : [α]λ= θ /(cl). An [α] in degrees would indicate the expected optical rotation for a 1 g/ml solution in a 10 cm = 1 dm cell. (Values are usually quoted at 20C).
A plot of optical rotation θ versus concentration should be linear and this is the basis for the use of polarimetry for quantitative analysis.
The analytical perfomance of the instrument was tested using a series of sucrose solutions made up in six different cells, each with a nominal path length of 10 cm and covering the concentration range from 0 to 10 wt%.
The same set of solutions was used to record readings with IR, red, yellow, green, blue and violet LED’s in the light source end of the instrument.
The data sets and calibration curves obtained from these measurements are shown opposite. The quality of the results is excellent, with R2 values in the range 0.9990-0.9996.
Traditionally, optical rotation measurements have been made using a Na lamp emitting at 589 nm. Note the higher sensitivity that is possible for sucrose detection by operating the polarimeter at a shorter wavelength.
Sucrose HydrolysisThe plot at left shows results obtained in a sucrose acid hydrolysis experiment. Here, 4 ml of concentrated HCl are rapidly mixed with 21 ml of a 40 wt/vol % solution of sucrose and the acidified solution is then quickly transferred to the polarimeter cell after data logging has commenced. Optical rotation values are recorded at ~3 second intervals over a one hour period.
Initially the optical rotation is positive, but as the sucrose hydrolyzes to an equi-molar mixture of glucose and fructose, the rotations decrease. D-fructose has a large negative specific rotation ([α]D=-89.50) while the value for D-glucose is positive ([α]D=+52.70). (The subscript D here refers to use of the Na D line at 589 nm).
As a consequence of these values the optical rotation undergoes sign inversion during the run. When the reaction goes to completion the 1:1 final mixture of the two sugars is sometimes referred to as “invert sugar”.
The rate of sucrose hydrolysis can be found by analyzing the data according to first order kinetics. An exponential fit to the optical rotation data is also shown at left; note the excellent agreement between the model fit and experiment.
Glucose MutarotationGlucose can exist in both open chain and cyclic structures, with the latter having two anomeric forms known as α-glucose (trans) and β-glucose (cis), differing only in the relative positioning of the hydroxyl group at the C-1 position and the -CH2OH group at C-5. Inter-conversion between these two forms in solution is known as mutarotation, with a change in optical rotation accompanying this process on account of the different specific rotations of the two anomeric forms : α(+112.20) and β(+18.70).
To study this process 5g of crystalline α-D-glucose and 0.5 ml of 2M H2SO4 are placed in a 50 ml volumetric flask that is quickly made up to the mark with distilled water. The resulting solution is immediately transferred to a polarimeter cell and the optical rotation monitored over a period of several hours. Representative results (experimental rotations and fit) are shown at left. The optical rotation decreases as the α-anomer converts to the β-anomer as equilibrium is approached.
The inset shows some additional data in which the acid dependence of the mutarotation rate has been investigated.
Wavelength Dependence of Optical Rotation : Sucrose, Glucose & Fructose
Optical rotation is wavelength dependent. This is clearly evident in the sucrose calibration curve experiments described earlier.
The specific rotation of a sample and the measurement wavelength λ are related by the Drude expression, [α]= A/(λ2-λ02). Here, two enantiomer-specific parameters - A and λ0 permit the sample’s specific rotation (and hence the observed rotation) to be predicted at any wavelength.
To demonstrate the validity of the Drude relationship, a high brightness 1W Luxeon white LED was placed in front of the entrance slit of a 1/8 m Oriel grating monochromator with manual wavelength tuning. The exit slit was removed from the monochromator and the emergent beam was then allowed to pass through the same aperture where the LED had previously been located.
The wavelength of the light passing through the sample cell could be varied over the wavelength region from 400 nm to 700 nm. Optical rotation measurements were then made at 10 nm intervals for solutions of known concentration for each of the three sugars : sucrose, fructose and glucose.
The Drude parameters A and λ0 for each sugar can be obtained from the slope and intercept of a plot of (1/θ) vs λ2, which should be linear. The plots of the results for each of the three sugars confirm this relationship.