Page 25 - LC-SFC_Pharma_Brochure

2. Column Dimensions and
2. Column Dimensions and Improving the speed of an existing analysis leads to higher overall ef-
Analytical Conditions
Analytical Conditions ficiency. For example, although quality control tests may be per-
formed according to the test methods registered with regulatory au-
UHPLC analysis generally requires a column that is shorter, has small- thorities, process control tests may not be under the influence of such
er internal diameter, and smaller packing material size. However, regulatory controls. In such cases, improving the speed of analysis
once the column dimensions are modified, the existing analytical during process control facilitates higher overall efficiency.
method must also be altered. This is because analytical conditions op-
timized for the HPLC analysis and the corresponding HPLC columns (a)
will not be optimal for the UHPLC analysis. The same is true when
transferring a method from UHPLC to HPLC. Prominence-i
When a method is transferred, the main items that need to be
changed are as follows. 5
2
• Flow rate 1
3 7
• Time program 4
• System delay volume 9 10
• Sampling rate 6 11
8
• Detector response
• Sample injection volume
0.0 10.0 20.0 30.0 40.0 min
• Upper pressure limit
ů Peaks
Among these parameters, the time program is particularly important 1. Cefadroxil, 2. Cefapirin, 3. Cefalexin, 4. Cefradine, 5. Cefotaxime, 6. Cefazolin,
for gradient analyses. If an inappropriate gradient program is config- 7. Cefuroxime, 8. Cefoxitin, 9. Cefoperazone, 10. Cefalothin, 11. Cefamandole.
Analytical Conditions
ured, it will result in an inferior separation after method transfer (see ů Column : Shim-pack VP-ODS (250 mm L. × 4.6 mm I.D., 5.0 µm)
Fig. 1). Further, if the relative retention times and resolution are speci- Mobile phase A : 0.1 % formic acid
fied in testing regulations, the time program must be modified to Mobile phase B : Acetonitrile
keep the separation pattern. Due to these complications, it is much Initial concentration : 5% B
Flowrate
: 1.0 mL/min
more efficient to modify a gradient program using theoretical calcula- Temp. : 45 °C
tions based on flow rate, column length, column internal diameter Detection : Ultraviolet-visible absorbance detector (260 nm)
and system delay volume. Injection volume : 10 µL
Time program
Time Unit Func. Value
2.00 Pump B.Conc 5
50.00 Pump B.Conc 35
ransfer
3. Example of Method T
3. Example of Method Transfer 55.00 Pump B.Conc 35
55.10 Pump B.Conc 5
65.00 Controller Stop
We have discussed method transfers from HPLC to UHPLC and UHPLC Method transfer
to HPLC. We will now describe examples of method transfer among (speed improvement)
multiple systems including Shimadzu’s systems and other vendor’s.
(b)
5 Nexera-i
Impr
ovement in the Speed of
3-1.
3-1. Improvement in the Speed of 2 3
Analysis of Cephem Antibiotics 1 4 7
Analysis of Cephem Antibiotics
Cephem antibiotics are beta-lactam antibiotics that can be adminis-
tered as an oral or injectable formulation. Here, we describe improv- 9
ing the analysis speed using a mixture of 11 cephem antibiotics as 6 10
samples. 8 11
We used a Shimadzu Prominence-i system to analyze cephem antibi-
otics under HPLC conditions (Fig. 2a). Based on the column dimen- 0.0 2.0 4.0 6.0 8.0 10.0 12.0 min
sions, we then modified the HPLC method such as the flowrate, ů Analytical Conditions
sample injection volume, and time program for UHPLC analysis (see Column : Shim-pack XR-ODS II (100 mm L. × 3.0 mm I.D., 2.2 µm)
Fig. 2 for details). We used the UHPLC method to perform an analysis Flowrate : 1.00 mL/min
with a Shimadzu Nexera-i system. As seen from the results shown in Injection volume : 4 µL
Time program
Fig. 2b, we succeeded in improving the speed of the analysis of the Time Unit Func. Value
cephem antibiotic mixture while keeping the separation pattern 0.51 Pump B.Conc 5
almost identical to that obtained from the HPLC analysis. The exam- 12.76 Pump B.Conc 35
35
B.Conc
14.04 Pump
ple shown in Fig. 1, which was a failed attempt at improving speed of 14.06 Pump B.Conc 5
analysis, also used cephem antibiotics as an analysis sample. These re- 17.00 Controller Stop
sults show that appropriate analytical conditions must be determined Note: Mobile phases A and B and initial concentrations are identical to those

before method transfer
during method transfer.
Fig. 2 An Example of Method Transfer (Cephem antibiotics)
2