manuf lab hrgr

• Direct analysis of samples might affect the accuracy of the method
• Preparation of pharmaceutical samples using different sample preparation methods

  1. Protein precipitation

  2. Liquid-liquid extraction

  3. Solid-liquid extraction

  4. Solid-phase extraction
• Sample components, if not removed, could destroy the analytical instrument
• Recoveries close to 100% are desirable
• The formula is used to measure recovery
• Recovery must be measured every time the method is calibrated with standards for quantitative analysis
• Pharmaceutical samples

  • Exist as mixtures of the analyte (target compound) and the sample matrix
  • Samples are seldom pure by nature
  • Crude plant extracts are composed of a wide assortment of secondary metabolites such as alkaloids, flavonoids, etc.
  • Proper sample preparation is imperative for accurate results
• Some analytes are very low in concentration in a mixture
• Recovery is the amount of the analyte retrieved after the sample preparation procedure
• Solid-Liquid Extraction of Aspirin from Aspirin Tablets

  1. Create a warm water bath by heating a large, half-full beaker of water on a heating stir plate

  2. Weigh ten aspirin tablets and record the mass exactly

  3. Crush the tablets into a fine powder in a mortar and pestle

  4. Transfer the powder to a 250-mL Erlenmeyer flask

  5. Use a graduated cylinder to collect 10 mL of absolute ethanol, pour it into the mortar and pestle, swirl to collect any remaining powder and pour the ethanol into the flask. Repeat the ethanol wash with another 10 ml

  6. Gently rest the Erlenmeyer flask of aspirin and ethanol in the warm water bath to increase the solubility of the acetylsalicylic acid. All the acetylsalicylic acid should dissolve within five minutes, while the other ingredients will not

  7. Set up a vacuum filtration apparatus, turn it on, place a filter paper in a filtration flask and connect the Büchner funnel. The acetylsalicylic acid in ethanol solution will pass through the filter paper, while the other ingredients should remain in the funnel

  8. Remove the ethanol by evaporation under low pressure. Remove the Büchner funnel from the flask and replace it with a rubber stopper

  9. Place the stoppered flask in the warm-water bath

  10. Turn the vacuum on and gently rotate or swirl the flask in the hot water bath

  11. Once the ethanol has evaporated, heat the flask by loosening the stopper

  12. Add 100 mL of distilled water to the flask and place it back in the warm-water bath. A temperature close to boiling works well

  13. If some of the acetylsalicylic acid remains undissolved, add another 10 mL of water

  14. Once all the acetylsalicylic acid has dissolved, decant it into a clean 250-mL beaker. A filmy substance on the surface of the water is fine

  15. Set up another Büchner funnel and filter on the filtration apparatus

  16. Decant the cold, settled acetylsalicylic acid/water mixture into the Büchner funnel, reserving the solids for last

  17. Add 5 ml of cold, deionized water to the beaker, swirl to collect remaining acetylsalicylic acid crystals and add to the Büchner funnel

  18. Repeat the cold-water wash three times

  19. Leave the flask and funnel on filtration to dry the crystals as much as possible, then break the vacuum and remove the funnel

  20. Cover with a watch glass and set aside to dry overnight

  21. Weigh the crystals and watch glass and record the mass exactly. Compute for the sample recovery
• Ethanolic Crude Extract

  • 9:1 Ethanol: Water
  • Aqueous Ethanolic Solution
  • Hexane fraction
  • Aqueous fraction
  • Ethyl Acetate fraction
  • Aqueous fraction
• Thin Layer Chromatography (TLC)

  In TLC, the adsorbent is spread in a thin layer over a solid support such as a sheet of aluminum (or glass) to make a TLC plate. Samples are spotted at the bottom of the plate which is developed by placing it in a chamber containing a small amount of the mobile phase. The mobile phase (a solvent) wicks up the plate by capillary action. TLC is used as an analytical technique and provides qualitative information about a sample. TLC is the most common and widely used method of analysis in a synthetic organic laboratory
• Liquid-Liquid Extraction of Flavonoids in Plant Extract

  1. Place the sample in iso sple layer tat and ad 50 mL of ethanol: water (9:1) mixture

  2. Stir for a while trying to dissolve as much sample as possible. Sonicate, if necessary

  3. The resulting solution is then transferred in a separatory funnel

  4. Add 50ml of hexane into the separatory funnel

  5. Transfer the remaining hexane layer to a 50 mL Erlenmeyer flask. Label and set this aside

  6. Place the aqueous layer back into the separatory funnel

  7. Add 50 ml of Ethyl acetate

  8. Repeat steps 6-7

  9. Transfer the aqueous layer (bottom) to a new Erlenmeyer flask and the ethyl acetate layer to a separate evaporating dish and allow the solvent to evaporate with gentle heating

  10. Determine the weight of the dried plant solvent fractions. Label it and reserve the samples for an upcoming lab exercise
• THIN LAYER CHROMATOGRAPHY

  1. Chromatography is the most versatile technique for separating mixtures

  2. Many different types or classes of chromatography exist and are used not only to separate, but also to isolate and identify analytes both in a qualitative and quantitative manner

  3. Chromatography employs a stationary phase and a mobile phase. Components of the mixture are carried past the stationary phase by the flow of the mobile phase. The components distribute, or partition, between the two phases, and separation occurs based on the average time a component spends in each of the phases

  4. Different classifications of chromatography are made based on the identity of each of the phases (for example, whether the mobile phase is a gas or a liquid) and the operating conditions of the system
• Purity test for Aspirin

  1. Preparation of the TLC Chamber

  2. Sample Application

  3. TLC Plate development
• Thin Layer Chromatography (TLC)


  • Most common and widely used method of analysis in a synthetic organic laboratory
  • Used due to low cost, rapid analysis time, convenience, and simplicity
• Phytochemical testing of Plant Solvent fractions

  Preparation of the TLC Chamber
• Sample Application

  1. Place samples in test tubes with methanol

  2. Draw lines on TLC plate and spot samples using capillary tube

  3. Develop the TLC plate in the chamber and mark the solvent front
• Preparation of the TLC Chamber

  1. Set up TLC chamber in a chemical fume hood

  2. Place filter paper in the tank to wick up solvent and saturate the tank with solvent vapor

  3. Pour ethyl acetate to create a layer no more than ½ cm deep and close the chamber to equilibrate
• Thin Layer Chromatography (TLC)

  Analytical technique providing qualitative information about a sample
• Rf value

  Number defined as distance traveled by component from application point divided by distance traveled by solvent from application point
• TLC Plate development

  1. Place the plate in the TLC chamber and close the lid

  2. Watch solvent travel upwards, mark the solvent front, allow plate to dry, visualize spots under UV lamp, measure distances traveled, calculate Rf values, and compare
• Spotting samples on TLC plate

  1. Immerse the capillary tube into the sample tube until some of the liquid is drawn into the capillary

  2. Very gently press the small end of the capillary tube at a crosshair on the TLC plate. Keep the spot small by touching the capillary quickly to the plate and then removing it immediately. Allow the spot to dry and repeat the spotting directly over your original spot
• Weighing and preparation of samples

  1. Weigh 5 mg of the (a) crude plant extract, (b) hexane solvent fraction, (c) ethyl acetate solvent fraction, and (d) aqueous solvent fraction

  2. Place the samples in separate 50 ml beakers

  3. Dissolve the samples in 2ml of Methanol
• Marginal utility is the additional utility (satisfaction) gained from the consumption of an additional product. If you add it up for each unit you get total utility
• Compound of Interest

  • Flavonoids/Steroids
  • Phenols Tannins Flavonoids
  • Alkaloids
• Phytochemical testing of Plant Solvent fractions

  1. Preparation of the TLC Chamber

  2. Weighing and preparation of samples

  3. TLC plate setup

  4. Spotting samples on TLC plate

  5. Developing the TLC plate

  6. Visualizing the spots

  7. Spraying with spray reagents

  8. Interpreting results
• Visualizing the spots

  Illuminate under the UV 254 and UV 366nm to visualize the spots
• Positive Result

  • Intense yellow to orange visible zones, Fluorescent color under 365nm
  • Blue spots
  • Brown-orange visible spots
• TLC plate setup

  1. Obtain a TLC plate. Handle the plate by the edges only, making sure to avoid touching the white adsorbent layer

  2. With a pencil (not pen), use a ruler to gently draw a line about 1 cm from the bottom of the plate. Be careful not to chip off the adsorbent layer
• Spray Reagent

  • Aluminum (III) chloride
  • Potassium ferricyanide-Ferric chloride
  • Dragendorff's reagent
• Developing the TLC plate

  1. Carefully place the plate inside the TLC chamber and immediately close the lid

  2. Watch carefully as the solvent travels upwards on the plate. Do not let the solvent rise until the tip of the plate. Remove the TLC plate and immediately mark where the solvent stopped rising with a pencil. This is called the solvent front

  3. Allow the TLC plate to dry
• Preparation of the TLC Chamber

  1. Begin by setting up two (2) TLC chambers (beaker covered with aluminum foil/watch glass) in a chemical fume hood

  2. Place a piece of filter paper upright in the tank to wick up solvent and saturate the tank with solvent vapor

  3. Prepare 30-ml of the following solvent mixture: (a) 5 parts chloroform: 1 part methanol, and (b) 100% ethyl acetate

  4. Pour in enough of solvent system A and solvent system B to create a layer no more than ½ cm deep and close the chambers. The tank needs to equilibrate
• Spraying with spray reagents

  Spray the TLC plates with the respective spray reagents to detect different phytochemical constituents