Page 42 - LC-SFC_Pharma_Brochure

2-2. Quantitation of a Mixture of Absorbance Normalized
255.93 nm
Two Components Spectrum of VP
Spectrum of DFBP
This section demonstrates that the derivative spectrum chromatogram
method can separate and quantitate two chromatographically co-
eluted peaks in a data set acquired for a mixture of two components.
Difluorobenzophenone (DFBP) and Valerophenone (VP) standards,
in 5 different relative concentrations of 100/1,100/10,100/50,
100/100,100/200, were used to acquire the derivative spectrum 216.93 nm
0
chromatograms of DFBP and VP. A calibration curve was created
(Table 1) and quantitative analysis of each sample mixture was 225.0 250.0 275.0 nm
Fig. 4 Spectrum comparison; DFBP and VP
performed (Tables 2 and 3).
Analytical Conditions
Table 1 Calibration data points created by derivative spectrum
Pump : Shimadzu LC-30AD×2 chromatogram of VP standard sample (R =0.9999309)
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Detector : Shimadzu SPD-M30A
Column oven : Shimadzu CTO-20AC Sample relative Retention
Controller : Shimadzu CBM-20A concentration time Area Concentration Error (%)
Autosampler : SIL-30ACMP (VP) (min)
Mobile phase : Acetonitrile45% / Water55%
Column : Shimadzu Shim-pack XR-C8 1 1.804 2,984 0.964 -3.62
(50 mmL. × 3.0 mmI.D., 2.2 µm)
Flow rate : 2 mL/min 10 1.801 30,368 9.876 -1.24
Column temp. : 40 °C 50 1.804 151,922 49.439 -1.12
Sampling : 80 msec
Slit width : 1 nm 100 1.802 310,801 101.149 1.15
Time constant : 240 msec
200 1.802 613,207 199.572 -0.21
Wavelength range : 190 nm to 700 nm
Injection volume : 1 µL
Fig. 4 shows the spectrum comparison of DFBP and VP. Fig. 5 shows Table 2 Quantitation result of VP in DFBP/VP mixed sample
the absorbance chromatogram of the mixed sample (DFBP/VP=
Sample relative Retention
100/200) at 210 nm and the derived spectrum chromatogram at concentration time Area Concentration Error (%)
st
st
255.93 nm (1 derived zero wavelength of DFBP) & 216.93 nm (1 (DFBP/VP) (min)
derived zero wavelength of VP). 100/1 1.808 3,167 1.023 2.30
Fig. 6 shows the absorbance chromatogram of the mixed sample 100/10 1.807 30,372 9.878 -1.22
(DFBP/VP=100/1) at 210 nm. Due to the low concentration in the 100/50 1.802 153,206 49.856 -0.29
sample, the VP peak is hidden in the DFBP peak. The ellipse in Fig. 100/100 1.806 309,596 100.757 0.76
6 shows the derivative spectrum chromatograms, which are used
100/200 1.815 620,556 201.964 0.98
by i-PDeA to find and integrate the peak.
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
Sample relative Retention
to calculate the quantitative amount of VP in each sample. The re- concentration time Area Concentration Error (%)
sults are shown in Table 2. In the case of the lowest VP concentra- (DFBP/VP) (min)
tion sample (DFBP/VP = 100/1), the concentration was calculated 100/1 1.746 359,670 102.225 2.23
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
100/100 1.742 357,891 101.719 1.72
results are shown in Table 3. Of note in these results is the repro-
100/200 1.751 351,528 99.911 -0.09
ducibility of peak area for a 1 µL sample injection (<1% RSD) as
Area %RSD=0.87 (injection volume:1 µL)
well as <3% error in the quantitative calculation.
mAU mAU
200 Ch1-210 nm, 4 nm VP 125 210 nm, 4 nm
Ch2-Derivative 255.93 nm Absorbance chromatogram
Ch3-Derivative 216.93 nm Absorbance 100
150 chromatogram
DFBP 75
Derivative spectrum
100
chromatogram
at 255.93 nm 50
Derivative spectrum
50 chromatogram 25
at 216.93 nm
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)
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