High Performance Liquid Chromatograph Nexera XR

Application Simple Labor-Saving Calibration Curve Creation

News
Using Autosampler Automatic Dilution Function
Part 2
Natsuki Iwata, Ryota Kanamaru, and Erika Baba

User Benefits
 The autosampler’s automatic dilution function reduces manual dilution preparation and organic solvent consumption.
 Simply specify the desired dilution ratio in the batch table, and use the same method file to automatically dilute solutions and
create a calibration curve.
 Setting and management are easy when changing HPLC conditions since a single method file is used regardless of the dilution ratio.

 Introduction A volume corresponding to the dilution ratio is aspirated from

The dilution of standard and sample solutions for HPLC analysis the stock solution vial and dispensed with the diluent into an
is generally performed manually, using pipettes. However, such empty vial (mixing vial) previously set in the autosampler (final
work is labor-intensive and time-consuming. In recent years, volume is 100 μL in this example). The solution in the vial is
automation for the purpose of labor-saving is desired to mixed using the aspiration/dispensing function (pipetting).
improve work efficiency and productivity. When the organic Finally, a specific amount of the solution is aspirated and
solvent is a diluent, a large amount of solvent is consumed to injected into the column.
prepare standard solutions for calibration curves in volumetric Table 1 Pretreatment Program
flasks, but the volume of sample solution required for HPLC
Line Command
analysis is only a few tens of μL or less.
1 a3=100/a2
Using the automatic dilution function equipped with Nexera
Autosamplers, it is possible to prepare a sample diluted at a user- 2 n.drain
defined factor and introduce it directly into the analytical column. 3 disp 600.0,rs
The Application News 01-00717 describes a simple method for 4 d.rinse
creating calibration curves using ultrapure water as a diluent. This 5 vial.n a0,a1
article introduces the analysis using organic solvent as a diluent. 6 n.strk ns
7 aspir a3,ss
8 air.a 0.1,ss
 Pretreatment Program and Operation 9 d.rinse
Overview 10 vial.n rn,sn
A method file contains information such as LC parameters, 11 n.strk ns
analytical parameters, and the pretreatment program. The 12 disp 100.1,rs
pretreatment program can set various dilution ratios, such as a 13 mix 1,5,40,ss,35
100-fold dilution. In addition, when the program is used with 14 n.drain
the batch add-in (Fig. 2 on the next page), a single method file
15 disp 100.0,rs
can be used regardless of the dilution ratio, thereby preventing
16 d.rinse
human errors such as setup mistakes.
17 inj.p
The dilution factor and conditions related to the mixing process
are configured using the LabSolutions workstation. The setup 18 v.inj
window for the autosampler pretreatment is shown in Fig. 1. 19 wait 2.0
Pretreatment program commands are shown in Table 1. In this 20 goto f0
article, the rinse solution was used as a diluent. 21 end

Fig. 1 Setup Window for Autosampler Pretreatment Program

Application
News

 Setting up a Batch Table mAU

A batch add-in called the SIL pretreatment variable is pre- 14 ■Peaks 2 276 nm
13 1. Coumarin
applied to LabSolutions to populate the batch table with the 12 2. Cinnamaldehyde
location of the stock solution vial and any dilution ratio*1. Fig. 2 11
shows the SIL pretreatment variable setup window for the 10 1
9
batch table. Set the plate number (A0), vial number (A1), and 8
dilution ratio of the stock solution vial (A2) in columns A0-A2 of 7
the SIL pretreatment variables. Place empty vials for automatic 6
dilution (mixing) at the locations specified by the tray and vial 5
4
numbers in the batch table. If the sample type (standard) and 3
level number are set as shown in Fig. 2, a calibration curve is 2
automatically generated after the analysis. Note that separate 1
analytical parameter settings are required. 0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min

*1 Contact Shimadzu for information about applying the batch add-in.

mAU
17.5 ■Peaks 2 288 nm
 Automatic Dilution Analysis for Mixed 15.0
1. Coumarin
2. Cinnamaldehyde
Standard Solution
12.5
An automatic dilution analysis was performed using a mixed
standard solution of coumarin and cinnamaldehyde at a 10.0
concentration of 100 mg/L each (prepared with acetonitrile). 1
7.5
Table 2 shows the analytical conditions. The pretreatment
program is the same as in Table 1. 1.0 mL polypropylene vials 5.0
were used for mixing, and septum vials were used for the stock
solutions (standard and sample). Acetonitrile was used as the 2.5
diluent for the rinse solution*2. Fig. 3 shows the chromatograms 0.0
of mixed standard solution (concentration after automatic
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min
dilution: 1.0 mg/L each) diluted 100-fold with acetonitrile.
Fig. 3 Chromatograms for Mixed Standard Solution of
Coumarin and Cinnamaldehyde
*2 For HPLC equipped with a multi-rinse function, use the rinse solution (Dilution Ratio: 100, Concentration after Automatic Dilution: 1.0 mg/L each)
specified in the parameter settings of an autosampler.

Table 2 Analytical Conditions

System: Nexera XR
Column: Shim-pack GIST-HP C18*3
 Repeatability
(150 mm × 3.0 mm I.D., 3 µm) The mixed standard solution (Dilution Ratio: 500, the
Flowrate: 0.8 mL/min concentration after automatic dilution was 0.2 mg/L each) was
Mobile Phase: A) Water analyzed six times consecutively using the autosampler
B) Acetonitrile
Time Program:
automatic dilution function. The repeatability (%RSD) of the
50 % B (0-2.0 min)→60 % B (4.0 min)→
100 % B (4.1 - 5.0 min) →50 % B (5.1 - 5.5 min) retention time and the peak area are shown in Table 3.
Column Temp.: 40 °C
Sample: 100 mg/L Coumarin and Cinnamaldehyde in Table 3 Repeatability (%RSD) in Six Replicate Analyses
Acetonitrile
Injection Volume: 5 µL Compound Retention time Peak area
Needle Stroke: 45 mm
Vial for Mixing: Shimadzu Vial, LC, 1 mL, Polypropylene*4 Coumarin 0.09 1.46
Vial for Stock Solution SHIMADZU LabTotal for LC 1.5 mL, Glass*5
and Sample: Cinnamaldehyde 0.06 1.82
Diluent: Rinse solution (Acetonitrile)
Detection: Ch1 (Coumarin): 276 nm, Ch2
(Cinnamaldehyde): 288 nm (SPD-M40)
*3 P/N: 227-30040-05, *4 P/N: 228-31600-91, *5 P/N: 227-34001-01

Plate No. 
Vial No. 
Dilution ratio 

Fig. 2 Setup Window for Batch Table SIL Pretreatment Variables

Application
News

mAU
 Calibration Curve
■Peaks 2 276 nm
Three calibration curves created with automatic dilution 1. Coumarin
25.0
function for coumarin and cinnamaldehyde (concentration 2. Cinnamaldehyde
range of 0.1-10 mg/L each) were analyzed.
20.0
Excellent linearities with coefficients of determination (r2) of
0.999 or greater were obtained. Calibration curves are displayed 15.0
in Fig. 4.
10.0
Peak area (x100,000) Peak area (x100,000)
3.0 Coumarin 7.5 Cinnamaldehyde 5.0 1

2.0 5.0 0.0

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min
1.0 2.5
mAU
35.0 ■Peaks 2
0.0 288 nm
0.0 1. Coumarin
0.0 5.0 Conc. 0.0 5.0 Conc. 30.0
(mg/L) (mg/L) 2. Cinnamaldehyde
25.0
Fig. 4 Calibration Curves (n = 3)
20.0
 Analysis of Cinnamon
15.0
A Sample of commercial Cinnamomum cassia was used. The
pretreatment protocol is the same as the process up to filtration 10.0
in Fig.3 from Application News No. 01-00233. Note that samples
5.0 1
were manually diluted with acetonitrile at the final step of
pretreatment in No. 01-00233-EN, but the sample was 0.0
automatically diluted with an autosampler in this article. The 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 min
pretreatment protocol is shown in Fig. 5. Acetonitrile was used
as the extraction solvent. Lipids were removed using a Fig. 6 Chromatograms of Cinnamomum Cassia Obtained by Automatic Dilution
dispersive solid phase extraction (dSPE) cartridge (Merck Supel (Dilution Ratio: 100)
QuE Z-Sep+). The cartridge eliminates the need to carry out
conditioning before loading samples, which simplifies
operations. Table 4 Analytical Results (n = 3)

Fig. 6 shows chromatograms obtained by diluting Concentration (mg/L)*6

Compound
Cinnamomum cassia extracts 100-fold with acetonitrile using an Automatic Manual
automatic dilution function. The two target compounds were Coumarin 0.28 (0.72) 0.27 (0.38)
well separated from the contaminants. The analytical results Cinnamaldehyde 1.90 (1.08) 2.02 (0.59)
(concentration after automatic dilution) are shown in Table 4. *6 Numbers in parentheses indicate %RSD, n = 3.
Table 4 also shows the analytical results obtained when
standard solutions for calibration curves were prepared
manually, and the 100-fold dilution with acetonitrile of the  Conclusion
pretreated cinnamon extract was performed manually. By automatically preparing standard solutions for calibration
curves at any dilution ratio and analyzing them directly, it was
possible to create a calibration curve easily and accurately. In
addition, when performing the determination of an actual
sample, dilution could be performed automatically. It was
confirmed that automatic determination was possible with only
simple pretreatment. The calibration curve creation method
described in this article is expected to lead to labor-saving for
analysts and solvent-saving from a sustainability viewpoint.

Related Applications
1. Simultaneous Quantitative Analysis of Coumarin and
Cinnamaldehyde in Cinnamon Produced in Different Regions
Application News No. 01-00233
2. Simple Labor-Saving Calibration Curve Creation Using
Autosampler Automatic Dilution Function
Application News No. 01-00717

Fig. 5 Pretreatment Protocol Contact Shimadzu for information about applying batch add-ins.

Nexera, LabSolutions, Shim-pack, and SHIMADZU LabTotal are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries.

01-00807-EN First Edition: Nov. 2024

For Research Use Only. Not for use in diagnostic procedures.
This publication may contain references to products that are not available in your country. Please contact us to check the availability of these
products in your country.
The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu.
See http://www.shimadzu.com/about/trademarks/index.html for details.
Shimadzu Corporation Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not
they are used with trademark symbol “TM” or “”.
www.shimadzu.com/an/ Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own.
The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its
accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the
use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject
to change without notice.

© Shimadzu Corporation, 2024