Qualitative determination of a pharmaceutical formulation
based on High-performance liquid chromatography and thin
layer chromatography.
Tymo Zwakenberg S4326253; Aniek barbier; October 10, 2022
Aim and principle
Two different methods of chromatography being high-performance liquid chromatography (HPLC)
and thin layer chromatography (TLC) are suitable for the qualitative determination of a
pharmaceutical formulation. TLC is an example of planar chromatography where the stationary
phase is located in a plane. The mobile phase then moves upward because of the capillary forces. In
order to make sure this procedure succeeds, the dried plate should be placed vertically in a thin
layer of mobile phase (eluent). The compounds of interest have been added on the plate before it’s
placed within the eluent. HPLC on the other hand has its stationary phase located inside a tube or
capillary, which is called the column. For HPLC an external force is always needed to be present to
carry the eluent along the stationary phase. A schematic overview of both setups can be found in
figure 1 and 2 [6].
Figure 1 & 2: Schematic overview of HPLC and TLC setup [4 & 5].
In order to acquire more knowledge and experimental skills with HPLC and TLC, a qualitative
determination of a given compound will be executed. This will be done by determining the effect of
methanol, pH and TEA presence on retention, executing and obtaining chromatographs and by
executing a TLC run.
, Materials and methods
All HPLC measurements were executed and recorded as chromatographs with a manual Hitachi
Primaide 1000. Whereas a TLC plate and UV-lamp were used to execute and review the TLC results.
The analyzed pharmaceutical formulation was provided as a tablet with reference number 2. The
qualitative characteristics of this tablet will be determined. The setup for this TLC experiment can be
found in the lab manual at page 41. The TA holds the list which tells which pharmaceutical
formulation corresponds with which diuretic. For all the executed steps the lab manual and/or lab
journal can be constructed. However, a few deviations on the lab manual will be listed, alongside the
materials, chemicals and calculation methods used.
Deviations:
- 10 µl was injected into the injection valve instead of 30 µl for every analysed sample.
- The drug formulation preparation was diluted a 100x before it was injected and analyzed.
- Every participant in the experiment was responsible for 2 HPLC setups, instead of 4.
Chemicals:
- Sodium nitrate (84.99 g/mole).
- Triethylamine (TEA, 101.193 g/mole).
- Triamterene (253.26 g/mole).
- Hydrochlorothiazide (297.74 g/mole).
- Furosemide (330.74 g/mole).
- Methanol (either in 45% or 55% volume).
- Wash (methanol/UP-water 50:50 v/v%).
- UP-water.
- Extraction mixture (glacial acetic/methanol/UP water in 20:40:40 v/v%).
- TLC eluent (water/methanol/glacial acetic acid/ethylene chloride in 10:15:25:50 v/v/v/v).
- Reference solution triamterene (26.3 mg/100ml).
- Reference solution hydrochlorothiazide (25.9 mg/100ml).
- Reference solution furosemide (23.5 mg/100ml).
- Pharmaceutical formulation (tablet 2 with unknown characteristics, 0.1785 g).
- Thymol.
Materials
- 100 ml volumetric flasks.
- 10-500 ml beakers.
- Watch glass.
- Injection syringe.
- Filter & syringe.
- Hitachi Primaide 1000
- Weighing scale.
- TLC plate.
- Chamber glass.
- 20 µl capillary.
- Oven.
- Ultrasonification bath.
- Filter paper.
based on High-performance liquid chromatography and thin
layer chromatography.
Tymo Zwakenberg S4326253; Aniek barbier; October 10, 2022
Aim and principle
Two different methods of chromatography being high-performance liquid chromatography (HPLC)
and thin layer chromatography (TLC) are suitable for the qualitative determination of a
pharmaceutical formulation. TLC is an example of planar chromatography where the stationary
phase is located in a plane. The mobile phase then moves upward because of the capillary forces. In
order to make sure this procedure succeeds, the dried plate should be placed vertically in a thin
layer of mobile phase (eluent). The compounds of interest have been added on the plate before it’s
placed within the eluent. HPLC on the other hand has its stationary phase located inside a tube or
capillary, which is called the column. For HPLC an external force is always needed to be present to
carry the eluent along the stationary phase. A schematic overview of both setups can be found in
figure 1 and 2 [6].
Figure 1 & 2: Schematic overview of HPLC and TLC setup [4 & 5].
In order to acquire more knowledge and experimental skills with HPLC and TLC, a qualitative
determination of a given compound will be executed. This will be done by determining the effect of
methanol, pH and TEA presence on retention, executing and obtaining chromatographs and by
executing a TLC run.
, Materials and methods
All HPLC measurements were executed and recorded as chromatographs with a manual Hitachi
Primaide 1000. Whereas a TLC plate and UV-lamp were used to execute and review the TLC results.
The analyzed pharmaceutical formulation was provided as a tablet with reference number 2. The
qualitative characteristics of this tablet will be determined. The setup for this TLC experiment can be
found in the lab manual at page 41. The TA holds the list which tells which pharmaceutical
formulation corresponds with which diuretic. For all the executed steps the lab manual and/or lab
journal can be constructed. However, a few deviations on the lab manual will be listed, alongside the
materials, chemicals and calculation methods used.
Deviations:
- 10 µl was injected into the injection valve instead of 30 µl for every analysed sample.
- The drug formulation preparation was diluted a 100x before it was injected and analyzed.
- Every participant in the experiment was responsible for 2 HPLC setups, instead of 4.
Chemicals:
- Sodium nitrate (84.99 g/mole).
- Triethylamine (TEA, 101.193 g/mole).
- Triamterene (253.26 g/mole).
- Hydrochlorothiazide (297.74 g/mole).
- Furosemide (330.74 g/mole).
- Methanol (either in 45% or 55% volume).
- Wash (methanol/UP-water 50:50 v/v%).
- UP-water.
- Extraction mixture (glacial acetic/methanol/UP water in 20:40:40 v/v%).
- TLC eluent (water/methanol/glacial acetic acid/ethylene chloride in 10:15:25:50 v/v/v/v).
- Reference solution triamterene (26.3 mg/100ml).
- Reference solution hydrochlorothiazide (25.9 mg/100ml).
- Reference solution furosemide (23.5 mg/100ml).
- Pharmaceutical formulation (tablet 2 with unknown characteristics, 0.1785 g).
- Thymol.
Materials
- 100 ml volumetric flasks.
- 10-500 ml beakers.
- Watch glass.
- Injection syringe.
- Filter & syringe.
- Hitachi Primaide 1000
- Weighing scale.
- TLC plate.
- Chamber glass.
- 20 µl capillary.
- Oven.
- Ultrasonification bath.
- Filter paper.
