Page 13 - Shimadzu Journal vol.2 Issue1

Technical Report Technical Report

As sx‘(λy) and sy’(λx) are non-zero constants in equations (1) and Fig. 3 shows the absorbance chromatogram at 235 nm and
Fractional Determination of Co-eluted Compounds Using (2), it can be seen that the derivative spectrum chromatogram at derived spectrum chromatogram at 1 derivative zero wavelength
st
wavelength λx shows the elution profile for component y only, and under several types of analytical conditions.
a New Data Processing Method for Photodiode Array Detector the derivative spectrum chromatogram at wavelength λy shows In (a), the impurity is detected using i-PDeA, even though MN and
the elution profile for component x only. its impurity overlap chromatographically.
That is, the derivative spectrum chromatogram at wavelength λx In (b), MN and the impurity begin to separate
1SJODJQMF BOE 4VNNBSZ PG J 1%F" *OUFMMJHFOU 1FBL %FDPOWPMVUJPO "OBMZTJT can separate component y only, and the derivative spectrum chromatographically. By comparing the spectra at the apex of
chromatogram at wavelength λy can separate component x only. each peak, MN and the impurity can be confirmed to be different
Toshinobu Yanagisawa 1
compounds.
1-2. Impurity Detection In (c), MN and the impurity are completely separated
Abstract chromatographically. The derived spectrum chromatogram shows
i-PDeA can detect whether impurity components exist in addition no significant signal at the retention time of the MN peak, which
The i-PDeA derivative spectrum chromatogram method was developed as a new data processing technique for photodiode array detectors to the major component. This method can be applied when one shows the signal of the MN peak does not include a contribution
for HPLC. A derivative spectrum is created by performing differential processing on the UV-Vis absorption spectrum at each measurement major component is mixed with other impurity components. The from the impurity.
time. Plotting the derivative spectrum values at the specified wavelength against retention time creates a derivative spectrum 3D chromatogram S (t, λ) can be expressed as follows if the major
chromatogram that is able to separate co-eluted peaks. The high selectivity of the derivative spectrum chromatogram can detect component elution profile is denoted as px (t), the impurity As described in this example, whether impurity peaks are
unexpected impurities and quantitate the target component only, without effects from interfering components that elute simultaneously. chromatographically resolved or co-eluted with the main
This paper formulates the theory of the derivative spectrum chromatogram method into mathematical expressions and reports details of component elution profiles as py (t), pz (t)…, the major component component, i-PDeA easily detects their existence.
verification of the basic performance using standard samples. spectrum as sx (λ), and the impurity spectra as sy (λ), sz (λ)…

Keywords:PDA data processing, peak deconvolution, derivative spectrum chromatogram, Nexera X2, UHPLC S , (t ) λ = p x (t )s x (λ ) + p y (t )s y (λ ) + p z (t )s z (λ )... 110 mAU
Ch1-235 nm, 4 nm
100 Ch2-Derivative 241.81 nm Absorbance
chromatogram of MN
Then, the derivative spectrum chromatogram at wavelength λx at 90
1. Basic Theory of the Derivative which the major component derivative spectrum chromatogram 80
Spectrum Chromatogram Method sx'(λ) value becomes zero is given by 70
60
1-1. Separation of Two Component ∂S 50 Derivative spectrum
Co-eluted Peaks ∂λ (t ) = p y (t )s y ( ' λ x ) + p z (t )s z ( ' λ x ) + ... 40 chromatogram
λ =λ x 30
Fig. 1 shows the absorption spectra of two components (target 20
component x and y), and Fig. 2 shows the derivative spectra Absorbance Therefore, the derivative spectrum chromatogram at wavelength λx 10
differentiated along the wavelength axis. In Fig. 2, the derivative is Spectrum of Target eliminates the major component elution profile and expresses the 0
zero for component x in the derivative spectrum at wavelength λx and S ( λ ) component x elution profiles of the impurities besides the major component. 0.21 0.22 0.23 0.24 0.25 0.26 0.27 min
Spectrum of Target
zero for component y at wavelength λy. component y (a) Mobile phase : Acetnirile95% / Water5%
mAU
90
Denoting the spectrum for target component x as sx (λ) and the peak 2. Examples of Analysis Using Ch1-235 nm, 4 nm Absorbance
Ch2-Derivative 241.81 nm
profile as px (t), and similarly the spectrum for target component y as the i-PDeA Functions 80 chromatogram of MN
sy (λ) and the peak profile as py (t), the 3D chromatogram S (t,λ) for 70
the two-component system in which component x and y both elute 2-1. Impurity Detection in 60 Impurity
can be expressed as: Standard Samples 50
Derivative spectrum MN
40
S , (t ) λ = p x t ) ( s x (λ ) + p y t ) ( s y (λ ) This section demonstrates that an impurity in a methylnaphthalene chromatogram
(MN) standard was detected using the derivative spectrum 30 Spectra of MN and impurity
Partial differentiation at wavelength λ gives the derivative spectrum chromatogram method. 20
chromatogram at wavelength λd as: Wavelength λ 10
Fig. 1 Spectra of two components
S ∂ (t ) = p (t )s ( ' λ ) + p (t )s ( ' λ ) Analytical Conditions 0
0.350
0.325
0.300
∂ λ λ = d λ x x d y y d (b) Mobile phase : Acetonitrile85% / Water15% min
As the derivative spectrum chromatogram at wavelength λx where the Derivative spectrum of Pump : Shimadzu LC-30AD×2 mAU
: Shimadzu SPD-M30A
Detector
Target component x 22.5 Ch1-235 nm, 4 nm
component x derivative becomes zero is dS ( λ ) Column oven : Shimadzu CTO-20AC 20.0 Ch2-Derivative 242.14 nm Absorbance
: Shimadzu CBM-20A
Controller
s x ( ' λ x ) = 0 d λ Derivative spectrum of Autosampler : Shimadzu SIL-30ACMP 17.5 chromatogram of MN
Target component y
we get, Column : Shimadzu Shim-pack XR-ODS 15.0
(30 mmL. × 3.0 mmI.D., 2.2 µm)
S ∂ (t ) = p (t )s ( ' λ ) ------ (1) Flow rate : 1 mL/min 12.5
∂ λ λ = x λ y y x 0 Column temp. : 40 °C 10.0 Derivative spectrum
: 80 msec
Sampling
Similarly, as the derivative spectrum chromatogram at wavelength λy Slit width : 1 nm 7.5 chromatogram
Time constant : 240 msec
where the component y derivative becomes zero is Wavelength range : 190 nm to 700 nm 5.0
s y ( ' λ y ) = 0 Injection volume : 1 µL 2.5
0.0
we get,
S ∂ (t p (t )s 1.6 1.7 1.8 1.9 2.0 2.1 min
(c) Mobile phase : Acetnitrile50% / Water50%
∂ λ λ = y λ ) = x x ( ' λ y ) ------ (2) λ x λ y Fig. 3 Absorbance chromatogram and derivative spectrum chromatogram
1. Analytical & Measuring Instruments Division Fig. 2 Derivative spectra of two components
39 40