Materials and Preparation: Preparation of calibration standards: 1. Add 1000ug/mL stock solutions of each of the tetracy
Posted: Wed May 04, 2022 3:33 pm
Materials
and Preparation:
Preparation of calibration standards:
1. Add 1000ug/mL stock solutions of each of the tetracyclines
into a wide-bottom, 1-mL volumetric flask according to the table
below to make five calibration solutions. Use methanol to dilute to
the mark.
Pre-treatment of the SPE cartridges:
1. Place three cartridges on the Visiprep manifolds securely.
Make sure that each one is attached to a new, clean liner that is
received by a test tube in the manifolds.
2. Add 2.0mL methanol to the cartridges, turn on the vacuum
valve and let drain. When the vacuum valve is fully open the flow
rate is approximately 2-mL/min.
3. Discard the drained solutions and return the test tubes to
the original positions.
4. Add 2.0mL McILvaine buffer solution into the cartridges and
let drain in the Visiprep manifolds in the same manner.
Preparation of the milk sample; SPE adsorption and
desorption of the milk sample:
8. Analyze immediately on the HPLC or keep
refrigerated in the dark. Shelf time should be kept to as short as
possible.
Calibration Curve and Data Handling
A calibration curve was generated using serial dilutions of a
1000 μg/mL OTC stock solution to final concentrations ranging from
20 to 100 μg/mL. Automatic peak integration was performed by the
computer software standard to the Shimadzu HPLC instrument.
Chromatograph peak areas obtained by two integration methods,
perpendicular-drop (PP) and valley-to- valley (VV), may both be
used for peak integration to illustrate the effects of software
choice on analytical results. The final OTC concentration in the
milk sample was calculated from the calibration curve, corrected by
a preconcentration factor of 2, and reported in micrograms per
milliliter.
This experiment takes between 4 to 8 h to complete depending on
how much material and instrumental preparation students have to
perform independently.
All chromatograms shown here were integrated by the automatic
integration program that is standard to the Shimadzu LC-20A HPLC
instrument. The HPLC chromatograms obtained with the calibration
standards are given in Figure 2 where the OTC elutes at
approximately 4.9 min. A molecular absorption spectrum obtained at
this elution time is shown in Figure 3, highlighting the advantage
of the PDA detector that enables the students to view a full scan
spectrum from 250 to 400 nm of the eluent, rather than a single
signal intensity at a set wavelength. A typical chromatogram
obtained from an extracted milk sample is shown in Figure 4, where
the purity demonstrated in the chromatogram indicates the
effectiveness of the SPE cleanup step. Representative calibration
curves using both PP and VV peak integration methods (Figure 5)
indicate excellent linearity by either data handling methods.
Typical students’ results from analyzing OTC recovered from the
spiked milk sample are listed in Table 2. For eight independently
prepared samples, the VV method generates a mean OTC concentration
of 49.4 μg/mL with 17.6% RSD and −1% relative error; the VV method
generates a mean OTC concentration of 53.8 μg/mL with 18.5% RSD and
8% relative error. A Student t test reveals that there is no
statistically significant difference between the two data-handling
methods at 95% confidence level.
Question: Make an estimate of how some of the results
were obtained in the table 2.
Volume of 1000-ug/mL standards added (UL) 20 40 60 80 100 Final concentrations of tetracycline (ug/mL) 20 40 60 80 100
Area of t = 4.9 min Peak (Arb Unit) 4 x 106 3 x 106 ■ valley-to-valley integration perpendicular- drop integration y = 1.438 x 106x + 4.172 × 105 R² = 0.9999 2x106 1x 106 1.722 x 105 y = 1.354 x 10 x R² = 0.9997 0 x 106 0.0 0.5 1.0 1.5 2.0 2.5 Mass OTC Injected Into Sample Loop / µg Figure 5. Typical calibration curves for OTC standards using perpendicular-drop and valley-to-valley baseline methods of integra- tion. -
Table 2. Student Results for the Analysis of Oxytetracycline in Spiked Milk Oxytetracycline in Milk/(mg/mL)ª Sample Perpendicular-Drop (PP) Valley-to-Valley (VV) 60.5 67.6 52.2 50.0 55.7 64.4 54.7 58.8 52.4 57.6 45.8 49.9 7 36.4 40.9 8 37.5 41.3 Mean 49.4 53.8 RSD (%) 17.6 18.5 "Each sample is spiked with 50 µg/mL OTC. 1 2 3 4 +56
and Preparation:
Preparation of calibration standards:
1. Add 1000ug/mL stock solutions of each of the tetracyclines
into a wide-bottom, 1-mL volumetric flask according to the table
below to make five calibration solutions. Use methanol to dilute to
the mark.
- Materials And Preparation Preparation Of Calibration Standards 1 Add 1000ug Ml Stock Solutions Of Each Of The Tetracy 1 (34.59 KiB) Viewed 32 times
1. Place three cartridges on the Visiprep manifolds securely.
Make sure that each one is attached to a new, clean liner that is
received by a test tube in the manifolds.
2. Add 2.0mL methanol to the cartridges, turn on the vacuum
valve and let drain. When the vacuum valve is fully open the flow
rate is approximately 2-mL/min.
3. Discard the drained solutions and return the test tubes to
the original positions.
4. Add 2.0mL McILvaine buffer solution into the cartridges and
let drain in the Visiprep manifolds in the same manner.
Preparation of the milk sample; SPE adsorption and
desorption of the milk sample:
8. Analyze immediately on the HPLC or keep
refrigerated in the dark. Shelf time should be kept to as short as
possible.
Calibration Curve and Data Handling
A calibration curve was generated using serial dilutions of a
1000 μg/mL OTC stock solution to final concentrations ranging from
20 to 100 μg/mL. Automatic peak integration was performed by the
computer software standard to the Shimadzu HPLC instrument.
Chromatograph peak areas obtained by two integration methods,
perpendicular-drop (PP) and valley-to- valley (VV), may both be
used for peak integration to illustrate the effects of software
choice on analytical results. The final OTC concentration in the
milk sample was calculated from the calibration curve, corrected by
a preconcentration factor of 2, and reported in micrograms per
milliliter.
This experiment takes between 4 to 8 h to complete depending on
how much material and instrumental preparation students have to
perform independently.
All chromatograms shown here were integrated by the automatic
integration program that is standard to the Shimadzu LC-20A HPLC
instrument. The HPLC chromatograms obtained with the calibration
standards are given in Figure 2 where the OTC elutes at
approximately 4.9 min. A molecular absorption spectrum obtained at
this elution time is shown in Figure 3, highlighting the advantage
of the PDA detector that enables the students to view a full scan
spectrum from 250 to 400 nm of the eluent, rather than a single
signal intensity at a set wavelength. A typical chromatogram
obtained from an extracted milk sample is shown in Figure 4, where
the purity demonstrated in the chromatogram indicates the
effectiveness of the SPE cleanup step. Representative calibration
curves using both PP and VV peak integration methods (Figure 5)
indicate excellent linearity by either data handling methods.
Typical students’ results from analyzing OTC recovered from the
spiked milk sample are listed in Table 2. For eight independently
prepared samples, the VV method generates a mean OTC concentration
of 49.4 μg/mL with 17.6% RSD and −1% relative error; the VV method
generates a mean OTC concentration of 53.8 μg/mL with 18.5% RSD and
8% relative error. A Student t test reveals that there is no
statistically significant difference between the two data-handling
methods at 95% confidence level.
- Materials And Preparation Preparation Of Calibration Standards 1 Add 1000ug Ml Stock Solutions Of Each Of The Tetracy 2 (233.47 KiB) Viewed 32 times
- Materials And Preparation Preparation Of Calibration Standards 1 Add 1000ug Ml Stock Solutions Of Each Of The Tetracy 3 (95.53 KiB) Viewed 32 times
were obtained in the table 2.
Volume of 1000-ug/mL standards added (UL) 20 40 60 80 100 Final concentrations of tetracycline (ug/mL) 20 40 60 80 100
Area of t = 4.9 min Peak (Arb Unit) 4 x 106 3 x 106 ■ valley-to-valley integration perpendicular- drop integration y = 1.438 x 106x + 4.172 × 105 R² = 0.9999 2x106 1x 106 1.722 x 105 y = 1.354 x 10 x R² = 0.9997 0 x 106 0.0 0.5 1.0 1.5 2.0 2.5 Mass OTC Injected Into Sample Loop / µg Figure 5. Typical calibration curves for OTC standards using perpendicular-drop and valley-to-valley baseline methods of integra- tion. -
Table 2. Student Results for the Analysis of Oxytetracycline in Spiked Milk Oxytetracycline in Milk/(mg/mL)ª Sample Perpendicular-Drop (PP) Valley-to-Valley (VV) 60.5 67.6 52.2 50.0 55.7 64.4 54.7 58.8 52.4 57.6 45.8 49.9 7 36.4 40.9 8 37.5 41.3 Mean 49.4 53.8 RSD (%) 17.6 18.5 "Each sample is spiked with 50 µg/mL OTC. 1 2 3 4 +56