Technical

C190-E240
Improving Peak Shape Using the Automatic

Technical Pretreatment Function (Co-Injection) in the
Report SIL-40 Series Autosamplers
Satoshi Akita1, Takato Uchikata1, Kenichi Yasunaga1, Hidetoshi Terada1, Takanari Hattori1, Keiko Matsumoto1
A b s tra c t:
If a sample solvent selected in accordance with the solubility of a compound has a higher elution strength than the mobile phase, this can have a
negative impact on the chromatogram. For example, if the ratio of organic solvent in a sample solvent is heightened in order to dissolve a
compound with low polarity, this can have a detrimental effect on peak shape for quickly eluted compounds in reverse-phase chromatography,
and even compromise the reliability of quantitative results. This article illustrates the use of the co-injection function, one aspect of the automatic
pretreatment functionality included as standard in autosamplers in the Nexera™ series (SIL-40 series), to improve peak shape by injecting a solvent
with a weak elution strength simultaneously with the sample in order to reduce the effect of the sample solvent. The co-injection function can
be specified easily via the LabSolutions workstation software.
Keywords: Automated pretreatment functionality, co-injection
1. Co-Injection Function It is also possible to successively aspirate user-defined volumes from

multiple vials for co-injection, as shown in Fig. 3.
In addition to regular injection procedures, autosamplers in the Nexera
In addition to co-injection, programmed pretreatment actions not
series (SIL-40 series) are equipped as standard with a variety of automatic
specified in a template can also be changed and executed.
pretreatment functions for diluting samples or adding reagents for exam-
ple. One such function, the co-injection function, can be used to take a
specified volume of reagent or solvent from a specified vial, and inject it
together with the sample, or to mix it with the sample inside the needle.
Using this function, a specified volume of reagent (or solvent) from a
specified vial can be injected together with the sample (Fig. 1).
(2)
(1)
(3)
(4)
(5)
(6)
Sample vial Co-injected solvent vial Injection port
Fig. 1 Illustration of the Injection Process

Fig. 2 Settings for Co-Injection Function
2. Settings for the Co-Injection Function

Settings for co-injection and other standard automatic pretreatment
functions can be configured easily using a LabSolutions template,
and saved in a method file (Fig. 2).
The applicable parameter settings are indicated below.
(1) Tray and vial number for the reagent to be co-injected
(2) Injection volume of reagent to be co-injected
(3) Injection timing (before, after, or before and after the sample)
(4) Mixing count and volume (number of agitation cycles within the
needle, and the volume)
(5) Wait time (wait time after mixing before injection)
(6) Air gap volume inserted before and after aspirating the sample
and the co-injected reagent
1 Analytical & Measuring Instruments Division Fig. 3 Settings for Co-Injection Function (Advanced)
3. Example of the Use of Co-Injection Fig. 5 shows the relationship between the quantity of co-injected
solvent (water) and the improvement in peak shape. The greater the
to Improve Peak Shape volume of co-injected water, the greater the improvement in the
The simultaneous analysis of lipid mediators and related substances is leading slope for both of the compound peaks, and in the resolution
mainly performed in the context of biomarker discovery and in disease (Rs) for both components.
research fields. Because lipid analysis involves the use of sample solvents Further improvements in peak shape and resolution can be expected
with a high organic solvent ratio, peak shapes for weakly retained com- from optimizing the type and volume of co-injected solvent.
ponents can become distorted during reverse-phase chromatography.
In this example, the co-injection function was used to improve the Tetranor-PGDM
peak shapes for a mixed standard solution prepared with 100 % Without co-injection
methanol as the solvent. Tetranor-PGEM
The effect of co-injecting water using the analytical conditions indi-

cated in Table 1 is shown in Fig. 4. Co-injection improved both the
peak shape and the tailing factor (S). It was particularly effective for
2.00 2.50 3.00 3.50 4.00 4.50 min
components with fast retention times.
Table 1 Analytical Conditions With 5 μL co-injected

Rs 2.05
Column :
C8 column (2.1 mm I.D. × 150 mm L, 2.6 μm)
Mobile Phase A :
0.1 % aqueous formic acid solution
Mobile Phase B :
Acetonitrile
Rinse Solution :
Acetonitrile
2.00 2.50 3.00 3.50 4.00 4.50 min
Other Conditions :
Compatibility with Shimadzu LC/MS/MS method package
for lipid mediators
Sample : Standard sample of lipid mediators and related compounds With 10 μL co-injected
Rs 2.34
(mixture of 196 components in methanol solution)
Co-Injected Solvent : Water (5, 10, and 15 μL)
Tetranor-PGFM With co-injection, 2.00 2.50 3.00 3.50 4.00 4.50 min
S: 1.07
Without co-injection,
S: 0.69
With 15 μL co-injected
Rs 2.41
1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 min
20-hydroxy-PGE2 With co-injection, 2.00 2.50 3.00 3.50 4.00 4.50 min
S: 1.07
Without co-injection, Fig. 5 Comparison of Resolution between Lipid Isomers
S: 0.71
(with 5 ,10 ,15 μL or no co-injection)
(Resolution Rs was calculated by the USP method.)
4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 min
2,3-dinor-11b-PGF2a With co-injection,

4. Summary
S: 1.02
Without co-injection, • Autosamplers in the SIL-40 series are equipped as standard with a
S: 0.95
function for automatically pretreating samples inside the au-
tosampler, including co-injection and dilution.
• For reverse-phase chromatography with samples that have a high
6.25 6.50 6.75 7.00 7.25 7.50 7.75 8.00 8.25 8.50 8.75 min
organic solvent ratio, the use of the co-injection function to simul-
Fig. 4 MRM Chromatograms for Various Lipids taneously inject a solvent with the sample can improve the shape
(with either 15 μL or no co-injection) of peaks with a short elution time, and improve the reliability of
(The tailing factor S was calculated by the USP method.) quantitative analysis results.
First Edition: August, 2019
© Shimadzu Corporation, 2019

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C190-E240

Improving Peak Shape Using the Automatic

Keywords: Automated pretreatment functionality, co-injection

1. Co-Injection Function It is also possible to successively aspirate user-defined volumes from

Sample vial Co-injected solvent vial Injection port

Fig. 1 Illustration of the Injection Process

2. Settings for the Co-Injection Function

methanol as the solvent. Tetranor-PGEM

The effect of co-injecting water using the analytical conditions indi-

Table 1 Analytical Conditions With 5 μL co-injected

2,3-dinor-11b-PGF2a With co-injection,

First Edition: August, 2019

© Shimadzu Corporation, 2019

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