Page 57 - LC-SFC_Pharma_Brochure

1400000
2
R =0.99998 5-3. Entries for Sample Solution
1200000
Analysis Dilution Template
1000000
Fig. 6 shows an example of template entries assuming auto setting
Peak Area 600000 of the sample stock solution vial position, and where the mixing vial
800000
(*2)
position is set to 15 vials offset
from sample stock vial position,
400000 and diluent consisting of rinse solution R0 is used to prepare a 1/10
200000 dilution for injection. As in the preparation of the standard
solutions, the final volume after dilution was set to 100 µL.
0
0 50 100 150 200 250 300
Concentration (mg/L)
Fig. 5 Caffeine Calibration Curve
Table 2 Repeatability Results of Standard Solution Repeat Analyses (n=6)
Concentration (mg/L) Area Value %RSD (%)
5 0.425
10 0.505
25 0.167
50 0.297
100 0.068 Fig. 6 Template Entry Example for Sample Solution Analysis
250 0.145 The requirements indicated in Fig. 6 are executed via the program
below.
Table 3 Dilution Accuracy at Each Dilution Ratio
n.drain : Needle is returned to the home position, then moved to the drain position,
and then slightly lowered.
Dilution Ratio (Factor) Dilution Accuracy (%) disp 600,rs : Liquid is dispensed at the set volume (600 µL) and speed (rs).
a0=sn+15 : Defines user variable (a0=sn+15).
2 100 vial.n rn,a0 : Needle moves to specified rack number (rn) and vial number (a0).
n.strk ns : Needle is lowered by the set needle stroke (ns) value.
5 100 aspir 10,ss : Liquid is aspirated at set volume
(post-dilution volume 100 µL / dilution factor 10 =10 µL) and speed (ss).
vial.n rn,sn : Needle moves to specified rack number (rn) and vial number (sn).
10 101 n.strk ns : Needle is lowered by the set needle stroke (ns) value.
disp 100,ss : Liquid is dispensed at the set volume (100 µL) and speed (ss).
20 101 a1=2 : Defines user variable (a1=2).
a2=10 : Defines user variable (a2=10).
50 101 mix 3,5,45,a1,a2 : Mixing is conducted the set number of times (3 times), with specified upper air volume
(5 µL), mixing volume (45 µL), aspiration speed (a1), and discharge speed (a2).
100 102 n.drain : Needle is returned to the home position, then moved to the drain position,
and then slightly lowered.
disp 100,rs : Liquid is dispensed at the set volume (100 µL) and speed (rs).
d.rinse : Needle moves to rinse port, and is immersed for the specified immersion time.
inj.p : Needle moves to injection port, and is lowered to bottom of injection port.
v.inj : High pressure valve is switched to injection side (flow line in which sample loop
connects with solvent delivery pump and column).
wait 1.0 : Operation waits until end of set wait time (1.0 min).
5. Analysis of Sample Solution goto f0 : Operation shifts to the set file number (standard injection operation f0).
end : Pretreatment program ends.
Here we describe the procedure for preparing a 1/10 dilution of the *2 Vial Offset Setting
sample stock solution using the SIL-30AC pretreatment feature. When the sample stock solution vial position is auto setting, this specifies the distance
between the injection vial (empty vial corresponding to sample number SN mixing occurs)
and sample stock solution vial as a vial offset number. In the example of Fig. 6 (offset 15),
this is defined as follows.
By entering (sample stock solution vial number) = (sample vial number) + 15 offset, it lets
5-1. Preparation Items the autosampler calculate the position of the stock and sample vials instead of the user
manually entering the numbers. This allows the same pretreatment file to be used with
different methods and batches with different numbers of samples, and also reduces errors if
1) Sample stock solution* (coffee, black tea, green tea) an incorrect vial number is set.
2) Vials for mixing (1.0 mL polypropylene vials) As shown in Fig. 7 below, if the vials containing sample (for example, No. 1−3) are spaced at
the same distance from the empty mixing vials (for example, 16−18), samples can be
* Filtered through membrane filter (0.2 µm pore size) pretreated and injected sequentially using a single pretreatment program with the variable
A0 used to increment vials. This vial position scenario is specified in the program using the
following expression:
Arrangement of vials is shown in Fig. 7.
5-2. Outline of Sample Solution
Analysis Operation Procedure To enter vial numbers for batch analysis or a single analysis,
specify the injection vial number (vial numbers 1−3).
The volume of sample stock solution corresponding to the specific
dilution ratio is aspirated from the stock solution vial, and is 1 Empty vial for mixing coffee (vial number 1)
2 Empty vial for mixing black tea (vial number 2)
dispensed into an empty vial along with diluent. Mixing is then
3 Empty vial for mixing green tea (vial number 3)
performed in the vial, and then the specified injection volume of
mixed solution is aspirated and introduced into the column. The 3 18 16 Coffee stock solution vial (vial number 16)
operation procedure is the same as that described above for analysis 217 17 Black tea stock solution vial (vial number 17)
18 Green tea stock solution vial (vial number 18)
of standard solution. 1 16
Fig. 7 Vial Arrangement Example
3