Unless otherwise stated in an individual monograph, 100 mg of a powdered botanical ingredient, 10 mg of a dry extract or fraction, or the amount of a dosage form containing the equivalent of the aforementioned quantities of the botanical ingredient is sonicated for 15 min with 1 mL of methanol. After centrifugation the filtrate or supernatant is used as the Sample solution. Unless otherwise stated in an individual monograph, 50 µL of an essential oil is dissolved in 1 mL of toluene and used as the Sample solution.

Unless otherwise stated in an individual monograph, USP Reference Standards of individual marker compounds are dissolved at a concentration of 1 mg/mL in methanol. The USP Reference Standard extracts are shaken and sonicated in methanol at a concentration of 10 mg/mL or, for essential oils, the USP Reference Materials are dissolved in toluene at a concentration of 50 µL/mL.

Samples are applied as narrow bands of 8.0 ± 0.5 mm length at a distance of 8.0 ± 0.5 mm from the lower edge of the plate. The system suitability standards are applied on the lane nearest to the edge at NLT 20 mm from the edge of the plate. The distance between tracks (center to center) is NLT 11.0 ± 0.5 mm. All application volumes are specified in the individual monograph. Application volumes usually range from 2.0 to 10.0 µL. The developing distance is marked with a pencil close to one of the edges of the plate before the development, although an electronic solvent front detection device may be substituted.

Following sample application and unless otherwise stated in an individual monograph, the plate is conditioned at a relative humidity of 33% for a minimum of 10 min (for example, by standing in a closed chamber containing a saturated solution of magnesium chloride).

Where a twin trough chamber is used, the rear trough is fitted with filter paper. The chamber is charged with a sufficient volume of developing solvent to wet the filter paper completely and achieve a level of developing solvent of exactly 5 mm in both troughs. With the lid closed, the chamber is left 20 min for saturation. The plate is introduced in a vertical position into the front trough of the chamber so that the coating layer faces the filter paper. When the mobile phase has reached a distance corresponding to a development path of 6 cm, the plate is removed from the chamber and dried in a vertical position in a stream of cold air that does not affect the integrity of the separated zones. Other chamber configurations and developing distances may be specified in an individual monograph. [Note—Other development chambers may be employed if the results obtained fulfill all of the system suitability criteria. ]

Where derivatization reagents are used, defined volumes of reagents in solution (typically 1–2 mL) are homogeneously sprayed onto the plate or the plate is immersed into the reagent solution at a defined speed and for a defined dwell time. [Note—Immersion speed of 50 mm/s and dwell time of 1 s works for most nonaqueous reagents. ]

Chromatograms on the plate are visualized as stated in an individual monograph. Observation and evaluation may be performed under UV 254 nm, UV 366 nm, or white light prior to and after derivatization.

To check the suitability of the system for resolution, position, and color of the bands, unless otherwise stated in an individual monograph, two or more reference substances are selected that have similar but just separable RF values under the chromatographic conditions to be used; for example, chlorogenic acid (blue) and hyperoside (yellow-orange) in chromatographic systems used for flavonoids. USP Reference Standard mixtures for system suitability may be provided, or the substances designated to check the system suitability for resolution, position, and colors of the bands may be included in the USP Reference Standard extracts. Description of the resolution, position, and colors for the key bands of the reference material fingerprint should match the description in the monograph within a specified tolerance range. The system suitability requirements in an individual monograph are satisfied when the results obtained comply with those specified in the monograph.

Chromatograms of the Sample solution and Standard solution are compared against the description in the Acceptance criteria section of the monograph with respect to zone position, zone separation, color, and relative intensity.

Documentation is necessary to record the results in an auditable manner to comply with current good manufacturing practices. Proper documentation tools should be employed; for example, a camera suitable for taking digital pictures under UV and white light and an imaging software suitable for archiving, retrieving, and analyzing the results makes it easy to maintain electronic records.