TLC Identification of Botanicals

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1.0 Purpose 
 
 The purpose of this procedure is to define (HP)TLC methods and conditions for the qualitative
 analysis of botanicals and their extracts in raw materials. 
 
 2.0 Scope 
 
 This procedure applies to the identification of all botanicals and complex materials. This SOP
 only applies to identification of raw materials using certified standards.
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC and analytical chemists to follow this procedure.
 
 3.2 It is the responsibility of QC Laboratory Management to implement this procedure and
 
 to ensure that the procedure is being followed. 
 
 3.3 It is the responsibility of Analytical Development to ensure that (HP)TLC is appropriate
 
 for ID. 
 
 3.4 It is the responsibility of QC Laboratory Management and/or Analytical Development
 
 to keep this procedure aligned with current practices. 
 
 4.0 Definitions 
 
 4.1 TLC — Thin Layer Chromatography 
 
 4.2 HPTLC — High Performance Thin Layer Chromatography 
 
 4.3 QC — Quality Control 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 TLC Identification of Botanicals D-742 3 Page 2 of 9 
 
 4.4 ID — Identification 
 
 5.0 References 
 
 5.1 D-742-F1, Form, TLC Botanical Identification Test Methods 
 
 5.2 D-742-F2, Form, TLC Developing Solution Recipes 
 
 6.0 Equipment and Materials 
 
 6.1 Supplies 
 
 6:1.1 Beaker — 500mL, 250mL, and 100mL 
 
 6.1.2 Whatman Filter Paper (4in diameter) 
 
 6.1.3 500mL Bottle with Cap 
 
 6.1.4 Pipettes — 10mL, ImL, and 10uL 
 
 6.1.5 Pipette Tips — 10mL, ImL, and 20uL 
 
 6.1.6 Petri Dish 
 
 Silica Gel 60 F254, 2.5cm x 7.5cm and S5cm x 10cm (HP)TLC Plates
 
 Scintillation Vials 
 
 6.1.9 2.0mL Eppendorf Tubes 
 
 6.1.10 Syringe — 3mL 
 
 6.1.11 Syringe Filters — Nylon and Polypropylene 
 
 6.1.12 Graphite Pencil 
 
 6.1.13 Capillary — 4” TLC Spotting Tubes 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 TLC Identification of Botanicals D-742 Page 3 of 9 
 
 6.1.14 Aluminum Foil 
 
 6.2 Equipment 
 
 6.2.1 Vacuum Oven 
 
 6.2.2 Cannon Rebel3 EOS Digital Camera 
 
 6.2.3 ChromaDoc-IT 125 Imaging System 
 
 6.2.4 Chromato-Vue C-75 UV Viewing Cabinet 
 
 6.2.5 Sonication Bath 
 
 6.2.6 Hot Plate 
 
 6.2.7 Fume Hood 
 
 6.2.8 Eppendorf Tube Rack 
 
 7.0 General Method 
 
 Note: Reference Form D-742-F1 Botanical Identification Test Methods for Test Conditions
 
 7.1 Chamber Preparation 
 
 7.1.1 Prepare a development chamber using a 250mL or 500mL beaker sealed with
 aluminum foil. 
 
 7.1.2 Fold a 4in diameter piece of Whatman filter paper or equivalent into a rack to
 support the plate and place into the covered beaker. Trim the paper as necessary
 
 to fit into the beaker. 
 
 7.1.3 Add 10mL or 15mL of mobile phase per D-742-F1 to the covered beaker.
 
 Equilibrate to room temperature. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 TLC Identification of Botanicals D-742 3 Page 4 of 9 
 
 7.2 TLC Plate Preparation 
 
 Tidak Draw a line ~1cm from the bottom of the plate with pencil, then place three hash
 
 marks, each ~8mm apart, to identify the lanes for a 2.5” x 7.5” plate. A line
 should be placed ~1cm from the top of the plate to mark the end point of the
 
 solvent front. 
 
 7.2.2 Each hash mark should be numbered from 1 to 3 beginning on the left.
 
 7.2.3 In the lem space at the top of the plate, the label information can be added. This
 
 includes the name of the botanical, the R number tested, and the date.
 
 7.2.4 Place the labeled (HP)TLC plate without sample into the chamber with mobile
 
 phase and allow the solvent front to migrate to the top of the plate.
 
 7.2.5 Dry the plate ~10 minutes on the hot plate in the fume hood.
 
 7.2.6 Allow the dried plate to cool. 
 
 7.3. Choosing a Mobile Phase 
 
 7.3.1 Choosing a solvent system as a mobile phase requires some knowledge of TLC.
 If all spots have an RF of 1, the mobile phase is too polar and its polarity needs
 
 to be reduced. If all spots have an RF of 0, then the mobile phase is too non-
 polar and the polarity needs to be increased. 
 
 7.3.2 Form D-742-F1 TLC Botanical Identification Test Methods has some
 | commonly used botanicals along with their mobile phases, dissolution media
 
 and developing solutions. 
 
 7.3.2.1 This should be used as a guide. Deviations from this are acceptable and
 should be noted on the write-up. 
 
 7.3.3 As needed, new materials will be compiled and added to Form F1 in groups.
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 TLC Identification of Botanicals D-742 3 Page 5 of 9 
 
 7.4 Sample Preparation 
 
 7.4.1 Pre-label the scintillation vials and Eppendorf tubes to identify the samples.
 
 7.4.2 Weigh 250mg of sample and transfer to the scintillation vial.
 
 7.4.3 Add 2.5mL of the appropriate dissolution solution per Form D-742-F1 to the
 
 vial and cap. 
 
 7.4.4 Sonicate both samples and standards for ~15 minutes in a sonication bath.
 
 7.4.5 Remove the samples from the bath and allow the samples to cool for ~10
 minutes. 
 
 7.4.6 Transfer supernatant into a pre-labeled Eppendorf tube. 
 
 7.4.7 Centrifuge for ~6 minutes at 5SOORPM in an Eppendorf microfuge.
 
 7.4.8 Transfer supernatant #2 to a fresh pre-labeled Eppendorf tube.
 
 TAS If necessary, the sample can be dried or concentrated in the vacuum oven
 
 (botanical test specific) or other method (like evaporation with nitrogen).
 
 7.5 Standard Preparation 
 
 7.5.1 If the standard in its whole form is suitable for the identification then use section
 
 7.3 for standard preparation. 
 
 7.5.2 If the standard in its whole form is not a suitable reference then refer to the
 
 manufacturer extract conditions per form D-742-Fl TLC _ Botanical
 Identification Test Methods of the raw material. Extract the standard under the
 
 same conditions to create a reference material. 
 
 
 

[SOP 

 Standard Operating Procedure | SOP No | Rev 
 TLC Identification of Botanicals D-742 3 Page 6 of9 
 
 7.5.3 As needed, general manufacturer extraction conditions for the raw material will
 
 be added to Form D-742-F1 TLC Botanical Identification Test Methods listed
 
 under the appropriate botanical. 
 
 7.6 Plate Spotting 
 
 7.6.1 Place the stenciled plate onto a hot plate pre-equilibrated to ~105°C.
 
 7.6.2 Some volatile components of a botanical sample or standard may not do well
 
 under heated conditions and may need to be air dried. 
 
 7.6.3 Using a capillary tube, spot the sample onto the plate slowly with small
 
 incremental volumes. Be sure to add spots directly on top of old spots. Offset
 
 spots will not give good results. Allow each addition to dry before adding the
 next increment. Keep spots < 2mm in diameter. Add appropriate volume of
 
 sample/standard to each lane as follows: 
 
 7.6.3.1 Lane 1: xuL Standard 
 
 7.6.3.2 Lane 2: xuL Sample A 
 
 7.6.3.3 Lane 3: xuL Standard or xuL Sample B 
 
 7.7 Plate Developing 
 
 7.7.1. Place cooled TLC plate into the chamber and cover. 
 
 7.7.2 Allow the solvent front to migrate to the top line then remove the plate from the
 
 chamber. 
 
 7.7.3. Heat dry the plate for approximately ten minutes before reading. This may be
 
 accomplished by placing onto a hot plate. Some volatile components of a
 botanical sample or standard may not do well under heated conditions and may
 
 need to be air dried. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 TLC Identification of Botanicals D-742 3 Page 7 of 9 
 
 7.8 Plate Reading 
 
 7.8.1 Plates should be evaluated visually for similarity between standard and test
 
 material during each interval of the plate reading process. 
 
 7.8.1.1 Visual assessment of the slide during each step in the development
 
 process is the primary means for determining the similarity between
 standard and test material. 
 
 7.8.1.2 Photographs and printouts of the plate are used for tracking purposes.
 
 7.8.2 Plates can be evaluated from the back (glass slide) of the plate if the band
 
 patterns are faint. 
 
 7.9 Photo Documentation 
 
 79.1 Take a digital picture in JPEG form of the TLC plate in ambient light using the
 
 <close> setting in the dial. 
 
 7.9.2 Place TLC into the viewing cabinet with the visible and UV lights on.
 
 7.9.3 Put the camera in <night> picture mode or <manual> shutter mode (for control
 of exposure time) using the dial. 
 
 7.9.4 Put the camera into manual focus mode by setting the switch to <MF>.
 
 7.9.5 Zoom the camera to maximize picture size by turning the zoom adjuster.
 
 7.9.6 Focus the camera manually by hand turning the focus adjuster until the image is
 clear and readable. 
 
 7.9.7 Set the UV to 365nm. 
 
 7.9.8 Turn off the visible light. 
 
 7.9.9 Take picture. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 TLC Identification of Botanicals D-742 3 | Page 8 of9 
 
 7.9.10 Switch the UV to 254nm. 
 
 7.9.11 Take picture. 
 
 7.9.12 If test conditions require adding a developing solution per form D-742-F2
 Developing Solution Recipes to enhance the picture, follow the instructions on
 
 forms D-742-F1 TLC Botanical Identification Test Methods and D-742-F2
 Developing Solution Recipes. 
 
 7.10 RF Value documentation 
 
 7.10.1 If required, RF Values can be documented for tracking purposes and easier
 
 comparison. 
 
 7.10.2 The Rf value is defined as the ratio of the distance moved by the solute and the
 distance moved by the solvent along the plate and is derived by the formula:
 
 7.10.3 RF values should be above 0.05 and below 0.95. Values of 0 or 1 should not be
 considered in determining matches between standards and samples.
 
 Distance component travelled 
 RF Value = 
 Distance solvent traveled (from spot location) 
 7.11 Picture Handling and Labeling 
 
 7.11.1 Pictures should be transferred from the SD card to a computer and archived in a
 
 dedicated folder. 
 
 7.11.2 Adjust contrast, rotate and crop the pictures as needed to maximize quality and
 
 readability. 
 
 7.11.3 Label the files as appropriate, indicating sample information and reading
 conditions. 
 
 7.12 Report Generation 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 D-742 3 Page 9 of 9 
 TLC Identification of Botanicals 
 
 7.12.1 An electronic report will be generated containing the following information:
 
 7.12.1.1. Name of Botanical (latin name) and part of plant 
 
 7.12.1.2 R#of Raw Material 
 
 7.12.1.3 Identification or Raw Material with extract ratio 
 
 7.12.1.4 Identification of each lane 
 
 7.12.1.5 Identification of each plate (exposure + developing conditions)
 
 7.12.1.6 A composite photograph (containing up to four different types of
 exposure + developing conditions) 
 
 7.12.1.7 Extraction conditions of raw material (if available)
 
 7.12.1.8 Standard used (vendor, part#, name, extract ratio, lot#) as necessary
 
 7.12.1.9 Ifrequired, record RF Values of major spots 
 
 8.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 02/04/14 | New \ 10/24/14 penta vom > op aan 2.5”x7.5” plate. Updated format. 14-0840 B. Johns | 14-0109 | B. Johns |

 3 | 02/28/23 | responsecstioin. bAliignledt wiyth curent practices coas-noss | K. Buetls
 
 
 

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