Page 18 - Shimadzu Journal vol.2 Issue1

Technical Report Technical Report

2. Examples of i-DReC Applications mAU 554 nm
New Data Processing Method for Photodiode 300
2-1. Extending the Linear Dynamic
Array Detector Range of Calibration Curves 200
This section demonstrates the extension of a calibration curve's
linearity into a high concentration range, using standard solutions
1SJODJQMF BOE 4VNNBSZ PG J %3F$ *OUFMMJHFOU %ZOBNJD 3BOHF &YUFOTJPO $BMDVMBUPS of Rhodamine with concentrations ranging from 0.01 g/L to 10 100
g/L. The following conditions were used for analysis.
Toshinobu Yanagisawa 1 347 nm
0
Analytical Conditions
200 300 400 500 600 nm
Abstract Pump : Shimadzu LC-30AD×2 Fig. 2 Spectrum of Rhodamine
: Shimadzu SPD-M30A
Detector
Column oven : Shimadzu CTO-20AC
A new data processing method for a photo diode array (PDA) detector, Intelligent Dynamic Range Extension Calculator (i-DReC) enables Controller : Shimadzu CBM-20Alite Area (×10 ) 6
the automatic calculation of peak area and height, utilizing spectrum similarity in the high concentration range where UV signal is Autosampler : Shimadzu SIL-30AC
saturated. When the integrated chromatographic peak area exceeds a user-defined threshold value, i-DReC automatically shifts the Mobile phase : Ammonium formate buffer 45% / ACN 55% 70
chromatographic profile to a wavelength with less UV absorption to prevent signal saturation. The absorption ratio between the original Column : Shimadzu Shim-pack VP-ODS
target wavelength and the wavelength used by the i-DReC function is applied as a correction factor to the peak area of the acquired (4.6 mmL. × 150 mmI.D., 5.0 µm) 60
chromatogram, thereby calculating the peak area and height at the original target wavelength. The i-DReC dramatically extends the linear Flow rate : 1 mL/min
Column temp.
: 40 °C
dynamic range of calibration curves, enabling reliable quantitation of high concentration samples without need for sample dilution and Sampling : 80 msec 50
reinjection, which would otherwise be required. Slit width : 1 nm
Time constant : 80 msec 40
Keywords:PDA data processing, dynamic range extension, Nexera X2, UHPLC Wavelength range : 190 nm-700 nm
Cell light path : 10 mm 30
Injection volume : 2 µL Calibration points lose linear
relationship in the high
20
1. Basic Principle of i-DReC 5. An absorption ratio (k) is calculated from the spectrum in (4). concentration area.
The intensity (Ia) of the spectrum at λa is divided by the intensity Fig. 2 shows the UV absorbance spectrum of Rhodamine, A 10
High concentration samples can produce saturated UV spectral Calibration curve was created based on peak area in the extracted
absorbance, which significantly affects peak area calculation and (Ib) of the spectrum at λb, as follows: chromatogram at 554 nm, the wavelength of maximum 0
causes a loss of linearity in the relationship of peak area to k=Ia/Ib absorbance, and is shown in Fig. 3a. At 1 g/L or greater 0 2.5 5.0 7.5 10
concentration. The i-DReC calculates an absorbance ratio between 6. Peak area and height of the measured peak in the concentration, the calibration curve exhibits the loss of linear Conc.(g/L)
the original target wavelength and another wavelength that chromatogram at λb are corrected by the absorption ratio to relationship between peak area and concentration. (a) Calibration curve at 554 nm
provides less absorbance in a spectrum on the down-slope of the determine the effective area and height at λa, as follows: Area (×10 ) 6
chromatographic peak where neither wavelength's absorbance is Peak area at λa = (peak area at λb) × k Fig. 3b shows the same calibration curve with i-DReC applied to
saturated. The corrected peak area and height are then calculated Peak height at λa = (peak height at λb) × k extend the linearity into the high concentration range. In this 250
by multiplying the measured peak area and height by the example, 347 nm was selected manually as the wavelength for Correlation factor
absorbance ratio. correction, and the spectrum used for sensitivity correction was 200 R=0.9999078
Extraction of chromatogram at λb extracted at an intensity of 700 mAU. The original peak area and the
mAU
Calculation Algorithm 4000 λa nm corrected peak area calculated by i-DReC is shown in Table 1. After
correction by i-DReC, the calibration curve based on the corrected
1. i-DRec is automatically applied when the intensity of a target 150
peak exceeds the user-defined threshold value. If the threshold peak areas exhibited excellent linearity with an unweighted
value is not exceeded, i-DReC is not applied. Threshold correlation factor of 0.9999078 and 0.9995750 weighted by 100
level
2
λb nm 1/(concentration) over the concentration range of 0.01 g/L to 10 g/L. Corrected by i-DReC
2. The wavelength used for correction by i-DReC (λb) can be set
either manually or automatically. When set manually, λb is a 0 Fig. 3C shows the error in concentration values obtained by inverse
user-defined parameter. When set automatically, λb is estimation using the i-DReC corrected calibration curve with 50
2
determined as follows: Extraction of weighting of 1/(concentration) . Even though the i-DReC corrected
spectrum for calibration curve extended the linear range of the original calibration
A UV spectrum is acquired at the retention time of the target peak. sensitivity correction curve by an order of magnitude, over the full range of concentration, 0
mAU 0 2.5 5.0 7.5 10
The spectrum is analyzed to determine an appropriate wavelength Signal intensity for Absorption ratio the error in calculated concentration value was within 5%. Conc.(g/L)
extraction of sensitivity
for which the absorbance is not saturated, which is then set as λb. correction spectrum is calculated by Ia (b) Linearity range extended by i-DReC
and Ib
3. The chromatogram at λb is extracted from the 3D data and Ia Table 1 Calibration points of Rhodamine samples Error (%)
correction
integrated to determine peak area and height. Ia Sensitivity k = Ib # Conc.(g/L) Peak area average (uAUsec) (n=2) 5
4. A UV spectrum is extracted from the chromatogram (at the spectrum 1 0.01 Original i-DReC
267,847
267,847
original target wavelength (λa)) at a point on the down-slope of 2 0.02 544,266 544,266 2.5
the peak (between the peak apex and peak end) where the λa 3 4 0.08 2,089,341 2,089,341 0
2,622,781
0.1
2,622,781
absorbance of neither λa nor λb are saturated. 5 0.2 5,255,999 5,255,999 0 2.5 5 7.5 10
Ib λb 6 0.5 12,072,748 12,282,271 -2.5
7 0.8 18,539,104 19,887,814
λ 8 1 21,823,608 24,644,792 -5
Fig. 1 Basic principle of i-DReC 9 2 33,708,885 49,250,552
10 5 53,883,445 126,813,723 (c) Error of corrected calibration points
11 8 65,182,276 198,990,013
12 10 71,500,307 245,336,353 Fig. 3 Calibration curve of Rhodamine
1. Analytical & Measuring Instruments Division
43 44