Thin Layer Chromatography TLC

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Thin Layer Chromatography (TLC)

• Rapid Screening of Pharmaceuticals by TLC
• Ross D. Kirchhoefer, trainer Allen Kenyon
  (deceased), Tom Layloff
• Gateway Analytical, 4041 Forest Park Ave., St.
  Louis, MO, 63108
• USP, Rockville, MD
• Kayla Laserson,Tom Kenyon
• CDC, Division of TB Elimination, Atlanta, GA
  30333/ CDC BOTUSA, Botswana

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Documentation cGMP/GLP (1)

• If it isnt written down, It didnt happen!
• Full description of standard, lot , purity, exp.
  date
• Full description of sample, dose, type of
  formulation, packaging etc.
• Full identification of chemicals, solvents, TLC
  test strips with lot , equipment, etc.
• Full description of reagent and/or mobile phase
  preparation.

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Documentation cGMP/GLP (2)

• Full description of sample and standard
  preparation.
• Formulas used for calculations and sample
  calculations.
• Describe experimental observations and include
  conclusions and/or results of the test.
• Use a notebook or worksheet for recording all raw
  data.
• Strikeouts must be initialed, dated and reason
  given for the strikeout.
• Test method must be referenced.

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Documentation cGMP/GLP (3)

• Original chromatograms (TLC) strips must be used
  for measurements and calculations (not copies).
• If data is maintained elsewhere, a reference must
  be made to its location.
• Complete documentation of analytical procedures
  and test results is a requirement of the FDC law.
  The analyst is responsible for his/her work.
• The USP PF lists the current USP reference
  standards.

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Objective

• To introduce the beginner to basic TLC principles
• To describe a simple and economical procedure for
  pharmaceutical screening

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Chromatography

• There are two basic types of chromatography
• Gas
• Liquid
• Liquid includes TLC and high performance liquid
  chromatography (HPLC)

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Introduction

• TLC is a form of liquid chromatography consisting
  of
• A mobile phase (developing solvent) and
• A stationary phase (a plate or strip coated with
  a form of silica gel)
• Analysis is performed on a flat surface under
  atmospheric pressure and room temperature

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Principles of TLC

• TLC is one of the simplest, fastest, easiest and
  least expensive of several chromatographic
  techniques used in qualitative and quantitative
  analysis to separate organic compounds
• Michael Tswett is credited as being the father of
  liquid chromatography. Tswett developed his ideas
  in the early 1900s.

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TLC

• The two most common classes of TLC are
• Normal phase
• Reversed phase

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Normal Phase

• Normal phase is the terminology used when the
  stationary phase is polar for example silica
  gel, and the mobile phase is an organic solvent
  or a mixture of organic solvents which is less
  polar than the stationary phase.

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Reversed Phase

• Reversed phase is the terminology used when the
  stationary phase is a silica bonded with an
  organic substrate such as a long chain aliphatic
  acid like C-18 and the mobile phase is a mixture
  of water and organic solvent which is more polar
  than the stationary phase.

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Adsorbents for TLC

• Silica gel
• Silica gel-F (Fluorescing indicator added)
• Magnesium Silicate (Florisil)
• Polyamides
• Starch
• Alumina

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Silica Gel (1)

• Silica gel is the most common adsorbent used in
  TLC
• It also comes with a fluorescing indicator added
  to it to make visualization or detection of
  sample spots easier

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Silica Gel (2)

• Silica gel is a polymer based on Silica to oxygen
  linkages with many -hydroxyl groups extending
  from this matrix
• Si-O-Si-O-Si-O-Si-O-Si-O-Si-(OH)x

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Silica Gel (3)

• They are very porous
• They have large surface area
• These affect your separation characteristics

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Silica Gel (4)

• The mode of separation is generally by adsorption
  or partition
• The more polar components will be adsorbed
  preferentially by the polar layer
• Hydrogen Bonding is the main force controlling
  adsorption between the silica gel surface and the
  analyte functional groups

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Steps in TLC Analysis

• The following are the important components of a
  typical TLC system
• Apparatus (developing chamber)
• Stationary phase layer and mobile phase
• Application of sample
• Development of the plate
• Detection of analyte

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General Procedure (1)

• Decide if you are going to do Normal or Reversed
  phase chromatography
• Prepare a plate or select a plate with the proper
  sorbent material
• Prepare the mobile phase
• Mark the plate
• Apply the sample
• Develop the plate
• Detect the analytes

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General Procedure (2)

• Silica gel with or without an added fluorescing
  indicator is the most commonly used and is
  classified as Normal phase chromatography
• The mobile phase is generally a non-polar solvent
  such as hexane. The hexane can be modified to a
  more polar solvent by the addition of or organic
  type solvents such as methanol, diethyl ether,
  ethyl acetate, toluene, dimethyl-formamide, etc.
  to achieve the required retention.
• The mobile phase can be further modified by the
  addition of acids or bases such as acetic acid or
  triethylamine to reduce tailing

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ProcedureTLC Plates

• The plates can be pre-marked for origin and
  development finish line as well as for sample
  zones
• Generally a distance of approximately 10 cm is
  used as the development of a plate so as to make
  the calculation of the Rf value easy.
• Rf is defined as the movement of the sample zone
  (x) divided by the movement of the developing
  solvent ( x/ 10 cm)

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ProcedureTLC Plate Development

• The development of the plate is linear and
  ascending
• The developing chamber is usually glass to
  prevent any interaction with the developing
  solvent and capable of holding the size plate you
  will be using
• The chamber may or may not be pre-saturated with
  the developing solvent
• Development may be with multiple solvents
• Development may be continuous (seldom used)
• Development may be two-directional (right angles)

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ProcedureDevelopment Chamber

• If a plate is placed in an unsaturated chamber,
  the air in the chamber is replaced by solvent
  molecules both from the evaporation of the
  solvent from the plate surface and the body of
  the developing solvent in the chamber
• If the developing solvent has more than one type
  of solvent, evaporation will be selective based
  on the boiling point of each solvent
• If a plate is placed in a pre-saturated chamber,
  no such evaporation can take place
• The separation and spot or zone shape may be
  different from these systems

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ProcedureSpot Movement

• The movement of the solvent up the plate is
  induced by capillary action
• Sample zone broadening always occurs and is
  caused by eddy and molecular diffusion as the
  spot moves up the plate
• The distribution coefficient of the sample solute
  affects the resolution of a separation i.e.
  solutes A and B
• the greater the distribution coefficient between
  solutes, the greater is the resolution between
  them

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Spotting the Sample

• The analyte must be in a suitable solution for
  spotting and any solvent can be used in other
  words the analyte must be in solution.

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Polarity

• Polarity of solutes Polar and non-Polar
• Polar solutes alcohols (ROH), acids (RCOOH),
  amines (RNH2)
• Polar solvents Methanol, ethanol, acetic acid
• Non-Polar solutes hydrocarbons, ketones
  (compared to methanol)
• Non-Polar solvents hexane, toluene (compared to
  methanol)

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Solvent Eulotropic Series

• Solvent E-value
• Toluene 0.29
• Chloroform 0.40
• Acetone 0.56
• Ethyl Acetate 0.58
• Ethanol 0.88
• Methanol 0.95
• Acetic Acid/Ammonia High
• Water High

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Calculation of Solvent Polarity

• Efinal xE1 xE2 ..xEn
• x volume fraction of solvent
• E E value of solvent
• Example
• 25 mL CHCL3 75 mL MeOH
• 0.25 x 0.40 0.75 x 0.88 0.76
  

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Like Dissolves Like

• Polar molecules favor polar solvents and vice
  versa
• Polar solutes migrate faster in polar mobile phase

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Performing the TLC AnalysisTypes of Materials
Needed

• Solvent bottles, 1 liter
• Small bottles, wide mouth 100 mL
• Graduated syringes, 1, 5 and 10 mL
• Pestle
• Graduated cylinders25, 50 and 100 mL
• Volumetric glassware and pipettes
• Small sample vials 1.5 amd 6 mL
• Micropipettes, 1,2,3,4 and 5 microliters
• Pasteur pipettes and rubber bulb, assorted sizes
• Beakers. assorted small sizes
• DiSPO test tubes 3 to 10 mL sizes

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Performing the TLC AnalysisProcedures

• Preparation of sample
• Preparation of standards
• Preparation of developing solvent (mobile phase)
• Plate marking
• Spotting a plate
• Placing plate in development chamber
• Conditioning development chamber
• Development of plate
• Visualization and interpretation
• Estimation of concentration
• Calculations of Rf values

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Performing the TLC AnalysisPreparation of Sample

• Take one dosage unit or a composite of dosage
  units and place in small plastic bag, grind to
  powder, or transfer to a suitable vessel and add
  proper solvent to dissolve active ingredient.
• Make stock and dilutions
• Stock solutions and dilutions must be calculated
• Example 5 mg / 5 mL
• Concentrations normally about 1 (one) mg/mL

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Performing the TLC AnalysisPreparation of
Standards

• Place one reference tablet into a vessel or a
  DiSPO test tube, add sufficient solvent to make a
  solution equivalent to 100 of the active dosage
  strength in the tablets (capsules) ie 5 mg / 5
  mL
• Dilute 4 parts of the 100 solution to 5 parts
  or alternately (take 4 parts of the 100 and add
  1 part solvent) this is an 80 value

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Performing the TLC analysis Making Your Own
Standards

• Use a reference standard tablets if available,
  or
• Alternatively use a primary or secondary standard
  which must be available. An amount is weighed on
  an analytical balance
• The proper dilutions are made to give the
  appropriate concentration equivalent to 100 of
  the active dosage strength in the prepared sample
  formulation
• 5 mg/ 5 mL
• The 80 standard is also prepared

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Performing the TLC Analysis Preparation of the
Mobile Phase

• The mobile phase (developer) is usually a mixture
  of solvents on a parts by volume basis
• One part chloroform and one part methanol would
  be noted as 11
• Pipettes or graduated cylinders can be used for
  these measurements
• Remember Polarity is controlled by your choice
  of solvents (see Eulotropic values)

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Performing the TLC AnalysisMarking the TLC Plate

• 5 x 10 cm plates or plastic backed strips should
  be provided in the kit
• If not, they can be cut from 20 x 20 cm plastic
  backed-silica coated sheets (but not recommended)
• Mark a line about 1 cm below the top
• Mark a small point on either side of your
  spotting point about 2 cm from the bottom
• Do not remove silica from sides, top or bottom
• Stay away from sides of cut plate when applying
  sample

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Performing the TLC AnalysisApplication of
Samples

• The plate is a plastic backed silica coated strip
• Spot 1 - 5 microliters of your sample in the
  center of the plate at the origin line
• Spot 1 - 5 microliters of the 100 standard and
  the 80 standard on either side of the sample
• Note If 3 microliters of sample is spotted,
  spot 3 microliters of the standards

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Performing the TLC AnalysisDry the Spots

• After spotting the sample and standards, the
  solvents must be evaporated from the spots before
  developing
• If aqueous solutions or partially aqueous
  solutions are used as solvents, several minutes
  may be needed to dry the spots

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Performing the TLC Analysis Assemble the TLC
Apparatus

• Assemble the TLC frame, plastic bag, saturator
  strips, aluminum holder, clamp and fishhook
• Add developing solvent
• If you wish to saturate the chamber, do so by use
  of the filter paper strip

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Performing the TLC AnalysisDevelopment of the
Plate

• Attach the TLC spotted plate (plastic
  backed-silica coated strip) to the aluminum frame
  with the clamp and lower into the plastic bag
  with the fishhook
• Allow the TLC plate to stay in the bag without it
  contacting the solvent for about 5 minutes to
  reach equilibrium
• Pull the plastic bag down to allow the developing
  solvent to contact the lower 1 cm of the TLC
  strip
• Develop the strip to the top marked line
• Stop the development
• Remove the TLC strip and allow the solvent vapors
  to evaporate

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Performing the TLC AnalysisVisualization and
Interpretation (1)

• Most pharmaceutically active drugs will not be
  visible to the naked eye
• Spots can be visualized by two basic techniques
• Ultraviolet light at 254 nm (shortwave UV). Long
  wave UV (340 nm) is used less commonly.
• Staining to make spots visible

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Performing the TLC Analysis Visualization and
Interpretation (2)

• Ultraviolet - short wave 254 nm
• Place the TLC strip under the UV light. Room
  light should be eliminated as much as possible
• If a silica gel F plate is used, the sample spots
  will appear as black spots on a fluorescent green
  background
• The sample zone intensity should be between the
  standards
• Battery operated shortwave UV lamps are
  available They are small and quite handy

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Performing the TLC AnalysisVisualization and
Interpretation (3)

• Staining
• Zones may be made visible by staining or spraying
  the TLC plate (strip) with a visualization
  reagent. Several different reagents can be used.
  Not all will visualize your sample zone.
• You must have the correct visualization reagent
  to be able to view your spots
• A universal visualization reagent is a 10
  sulfuric acid solution. When sprayed on your
  plate, the plate is heated and your spots are
  charred which can be seen by eye. Glass plate
  only. Several details may need to be worked out
  with this reagent

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Performing the TLC Analysis Visualization and
Interpretation (4)

• Staining
• Prepare the staining reagent and place in a
  plastic bag
• Dip the TLC strip into the reagent
• Remove the TLC strip and observe the spots
• The sample zone intensity should be between the
  standards

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Performing the TLC Analysis Calculate the Rf
Values

• The Rf value is calculated by measuring the
  distance the sample zone travels divided by the
  distance the developing solvent travels
• Values below 0.1 is considered poor the spots
  are too close to origin
• Values of 0.1 to 0.8 are good and any other spots
  (impurities) or other actives are resolved form
  each other
• Above 0.8 poor spots may be too broad or
  distorted

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Performing the TLC Analysis Acceptance or
Rejection Criteria

• Sample zone has an intensity between the
  standards of 80 - 100 ACCEPT
• If lower - REJECT
• If higher, re-spot a standard at a concentration
  higher than 100 , the 100 standard and the
  sample may be needed to estimate concentration
• Normally, bad drug samples will be lower than
  100 level and this is not a concern
• Sample zones should be resolved from any other
  active drugs (combination drug products like
  Isoniazid and Rifampin) ACCEPT
• Sample zones should be separated from any
  decomposition products, impurities or excipients
  in the drug formulation. If many alternate zones
  besides the active are found, the drug may be
  decomposed REJECT and do more testing

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Performing the TLC Analysis Acceptance or
Rejection Criteria

• There should be no additional zones is the
  sample ACCEPT
• There should be no unexplained zones in the
  sample ACCEPT
• The sample and standard should have identical Rf
  values ACCEPT
• If the sample and standard have different Rf
  values REJECT

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Development of a New TLC Method

• Determine the drug type , its polarity, and/or
  acidity or basicity.
• Find a solvent the drug is soluble to extract
  from the matrix
• Select a TLC strip or plate you feel may be
  suitable, ie silica gel or silica gel G-F240 may
  be a good first choice
• Prepare a mobile phase you feel may provide Rf
  values between 0.1 and 0.8
• Find a technique to visualize the drug
• Test
• If necessary, modify to improve the technique

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Advantages of TLC

• Low cost
• Short analysis time
• Ease of sample preparation
• All spots can be visualized
• Sample cleanup is seldom necessary
• Adaptable to most pharmaceuticals
• Uses small quantities of solvents
• Requires minimal training
• Reliable and quick
• Minimal amount of equipment is needed
• Densitometers can be used to increase accuracy of
  spot concentration

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TLC Problems Troubleshooting

• Over migration Developer too polar Reduce
  polarity
• Under migration Developer too non-polar Increase
  polarity
• Distorted solvent front Developer not
  equilibrated Equilibrate
• Distorted spots Wrong adsorbent Change plates
• Distorted spots Spotted too much Change
  concentration
• No separation Wrong developer Change developer
• No separation Wrong adsorbent Change plate type
• Tailing Spot overloading Reduce concentration
• Tailing Component is basic Increase acidity
• Tailing Component is acidic Increase basicity
• Tailing/no separation Decomposition Developer/pla
  te

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Kodak Slide Show

• Review of Safety
• Review of Performing the TLC Analysis
• Preparing the Sample alternatives given by
  instructor
• Preparing the standard
• Preparing the plate (silica gel strip)
• Marking the plate (strip)
• Spotting the sample
• Developing the plate, calculate Rf
• Visualization techniques

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