TLC Overview

8/8/2019 TLC Overview

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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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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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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 procedurefor 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 underatmospheric 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 ofliquid 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 theanalyte 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 sorbentmaterial

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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Procedure:

TLC Plates

The plates can be pre-marked for origin and developmentfinish line as well as for sample zones

Generally a distance of approximately 10 cm is used asthe development of a plate so as to make the calculationof 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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Procedure:

TLC 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 plateyou 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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Procedure:

Development Chamber

If a plate is placed in an unsaturated chamber, the air in thechamber is replaced by solvent molecules both from theevaporation 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 eachsolvent

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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Procedure:

Spot Movement

The movement of the solvent up the plate is induced bycapillary action

Sample zone broadening always occurs and is caused byeddy and molecular diffusion as the spot moves up theplate

The distribution coefficient of the sample solute affectsthe 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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LikeDissolves Like

Polar molecules favor polar solvents and vice

versa

Polar solutes migrate faster in polar mobile phase


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Performing the TLC Analysis:

Types 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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Procedures

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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Preparation 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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Preparation 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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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 1:1

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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Marking 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-silicacoated 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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Application 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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Dry the Spots

After spotting the sample and standards, the

solvents must be evaporated from the spots beforedeveloping

If aqueous solutions or partially aqueous solutions are

used as solvents, several minutes may be needed to

dry the spots


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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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Development 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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Visualization 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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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 aresmall and quite handy


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Staining

Zones may be made visible by staining or spraying the

TLC plate (strip) with a visualization reagent. Severaldifferent reagents can be used. Not all will visualize yoursample zone.

You must have the correct visualization reagent to beable to view your spots

A universal visualization reagent is a 10% sulfuric acid solution. Whensprayed on your plate, the plate is heated and your spots are charred whichcan be seen by eye. Glass plate only. Several details may need to be workedout with this reagent


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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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Calculate the Rf Values

The R f 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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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% standardand 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 arefound, the drug may be decomposed; REJECTand do more testing


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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 silicagel 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 equilibratedEquilibrate

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/plate


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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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TLC Overview

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