Page 15 - Shimadzu Journal vol.2 Issue1

Technical Report Technical Report

2-2. Quantitation of a Mixture of Absorbance Normalized 3. Summary of i-PDeA Settings 4. Conclusion
Two Components Spectrum of VP 255.93 nm The parameters for the i-PDeA function are set as part of the data The excellent performance and reproducibility of the SPD-M30A
Spectrum of DFBP
This section demonstrates that the derivative spectrum chromatogram processing method of the photodiode array detector. Once the photodiode array detector and the Nexera X2 system make this
method can separate and quantitate two chromatographically co-eluted analytical protocol is defined, it can be applied for routine new separation methodology possible. The i-PDeA function
peaks in a data set acquired for a mixture of two components. analysis. The following is a brief summary of the i-PDeA helps increase the speed of analysis and enhances laboratory
parameters. productivity. To summarize the key benefits:
Difluorobenzophenone (DFBP) and Valerophenone (VP) standards,
in 5 different relative concentrations of 100/1,100/10,100/50, 1. The Savitzky-Golay method is used to determine the first Co-eluted peaks can be separated mathematically, using
100/100,100/200, were used to acquire the derivative spectrum derivative spectrum, from which a list of wavelengths where derivative spectrum chromatograms
chromatograms of DFBP and VP. A calibration curve was created 216.93 nm st
0 the 1 derivative value is zero is generated. Spline interpolation Poorly resolved peaks are processed and visualized as pure
(Table 1) and quantitative analysis of each sample mixture was is applied to calculate the wavelength closest to the 1 peaks with no contribution from co-eluting components
st
performed (Tables 2 and 3). 225.0 250.0 275.0 nm derivative zero wavelengths, and the results are displayed in a
Fig. 4 Spectrum comparison; DFBP and VP Impurity peaks hidden by, or even in, the target peak can be
table. detected
Analytical Conditions Table 1 Calibration data points created by derivative spectrum
Pump : Shimadzu LC-30AD×2 chromatogram of VP standard sample (R =0.9999309) Fast and accurate quantitative analysis is possible, even
2
Detector : Shimadzu SPD-M30A without complete chromatographic separation
Column oven : Shimadzu CTO-20AC Sample relative Retention
Controller : Shimadzu CBM-20A concentration time Area Concentration Error (%) Simple post-run analysis procedure
Autosampler : SIL-30ACMP (VP) (min) The i-PDeA function provides a new solution, which is useful for
Mobile phase : Acetonitrile45% / Water55%
Column : Shimadzu Shim-pack XR-C8 1 1.804 2,984 0.964 -3.62 identification and quantitation of impurities. The use of this
(50 mmL. × 3.0 mmI.D., 2.2 µm) feature is expected to increase laboratory efficiency and
Flow rate : 2 mL/min 10 1.801 30,368 9.876 -1.24 produce more reliable analytical data.
Column temp. : 40 °C
Sampling : 80 msec 50 1.804 151,922 49.439 -1.12
Slit width : 1 nm 100 1.802 310,801 101.149 1.15 2. i-PDeA most effectively resolves co-eluted peaks when the peak
Time constant : 240 msec height in the derivative spectrum chromatogram for one References
Wavelength range : 190 nm to 700 nm 200 1.802 613,207 199.572 -0.21 st
Anal. Chim. Acta
Injection volume : 1 µL component, taken at a 1 derivative zero wavelength of another 1) A. Lober, , 164, 293-297 (1984)
component, is sufficiently large. If the shape of the spectra of 2) B. Kowalski ., ., 58, 496-499 (1986)
et al
Anal. Chem
Table 2 Quantitation result of VP in DFBP/VP mixed sample
two components is very similar, i-PDeA cannot be applied. 3) T. Ryan ., ., 16 (7) , 1545-1560 (1993)
et al
J. Liq. Chromatogr
Fig. 4 shows the spectrum comparison of DFBP and VP. Fig. 5 shows Sample relative Retention To extract the derivative spectrum chromatogram, plotting the 4) B. Vandeginse ., , 173, 153-164 (1985)
et al
the absorbance chromatogram of the mixed sample (DFBP/VP= concentration time Area Concentration Error (%) derivative spectrum values at the specified wavelength against Anal. Chim., Acta
et al Chemom. Intell. Lab. Sys
100/200) at 210 nm and the derived spectrum chromatogram at (DFBP/VP) (min) retention time, select “Derivative” for Chromatogram Type and 5) M. Maeder ., ., 3, 205-213 (1988)
et al
J. Of Chromatog
st
st
255.93 nm (1 derived zero wavelength of DFBP) & 216.93 nm (1 100/1 1.808 3,167 1.023 2.30 using the wavelength obtained by the Detect 1 Derivative Zero 6) I. Sakuma ., ., 506, 223-243 (1990)
st
Analyst
et al
derived zero wavelength of VP). 7) A. Yamamoto ., , 120, 377-380 (1995)
100/10 1.807 30,372 9.878 -1.22 function, set the value (with 2 decimal places) of the 8) T. Hakuta ., , 25, 1149-1153 (2009)
et al ANALYTICAL SCIENCE
Fig. 6 shows the absorbance chromatogram of the mixed sample 100/50 1.802 153,206 49.856 -0.29 Wavelength in the Wavelength Settings window of the
et al
J. Sep. Sci
(DFBP/VP=100/1) at 210 nm. Due to the low concentration in the Multi-Chromatogram table. 9) K. Uchiyama ., ., 34, 1525-1530 (2011)
sample, the VP peak is hidden in the DFBP peak. The ellipse in Fig. 100/100 1.806 309,596 100.757 0.76 10) S.Kazuhara, Food and Agricultural Materials Inspection
6 shows the derivative spectrum chromatograms, which are used 100/200 1.815 620,556 201.964 0.98 Center Research Report, 23, 77-86 (1999)
et al BUNSEKI KAGAKU
by i-PDeA to find and integrate the peak. 11) K. Uchiyama ., , 60 (2) , 171-174 (2011)
12) S. Kodama ., , 45 (3) , 259-263 (1996)
et al BUNSEKI KAGAKU
The VP calibration curve, created by using the integrated peak Table 3 Quantitation result of DFBP in DFBP/VP mixed sample
area for VP in the derivative spectrum chromatogram, was used to Sample relative Retention
calculate the quantitative amount of VP in each sample. The concentration time Area Concentration Error (%)
results are shown in Table 2. In the case of the lowest VP (DFBP/VP) (min)
concentration sample (DFBP/VP = 100/1), the concentration was 100/1 1.746 359,670 102.225 2.23
calculated to be 1.023 (2.30% error).
100/10 1.742 357,969 101.741 1.74
In the same way, the DFBP calibration curve was created and used 100/50 1.737 357,497 101.607 1.61
to calculate the quantitative amount of DFBP in each sample. The
results are shown in Table 3. Of note in these results is the 100/100 1.742 357,891 101.719 1.72
reproducibility of peak area for a 1 µL sample injection (<1% RSD) 100/200 1.751 351,528 99.911 -0.09 3. The derivative spectrum chromatogram has positive value when
as well as <3% error in the quantitative calculation. the slope of the spectrum is up and negative value when the
Area %RSD=0.87 (injection volume:1 µL) slope of the spectrum is down. Peak direction in the derivative
mAU mAU spectrum chromatogram can be adjusted by setting the
200 Ch1-210 nm, 4 nm VP 125 210 nm, 4 nm polarity, and peak size can be adjusted by setting the factor.
Ch2-Derivative 255.93 nm Absorbance chromatogram
Ch3-Derivative 216.93 nm Absorbance 100
150 chromatogram Set integration and quantitation parameters for the derivative
DFBP 75 spectrum chromatogram.
Derivative spectrum
100
chromatogram The derivative spectrum chromatogram can be handled the
at 255.93 nm Derivative spectrum 50 same as other multi-chromatograms for quantitative purposes.
50 chromatogram
at 216.93 nm 25
0
0
1.60 1.65 1.70 1.75 1.80 1.85 1.90 1.95 min 1.60 1.65 1.70 1.75 1.80 1.85 1.90 1.95 min
Fig. 5 Peak separation in the mixed sample (DFBP/VP=100/200) Fig. 6 Absorbance chromatogram of the mixed sample (DFBP/VP=100/1)
41 42