Date post: | 09-Apr-2018 |
Category: |
Documents |
Upload: | mahesh-deshpande |
View: | 220 times |
Download: | 0 times |
of 51
8/8/2019 TLC Overview
1/51
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
8/8/2019 TLC Overview
2/51
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.
8/8/2019 TLC Overview
3/51
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.
8/8/2019 TLC Overview
4/51
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.
8/8/2019 TLC Overview
5/51
Objective
To introduce the beginner to basic TLC principles
To describe a simple and economical procedurefor pharmaceutical screening
8/8/2019 TLC Overview
6/51
Chromatography
There are two basic types of chromatography
Gas
Liquid
Liquid includes TLC and high performance liquid
chromatography (HPLC)
8/8/2019 TLC Overview
7/51
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
8/8/2019 TLC Overview
8/51
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.
8/8/2019 TLC Overview
9/51
TLC
The two most common classes of TLC are:
Normal phase Reversed phase
8/8/2019 TLC Overview
10/51
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.
8/8/2019 TLC Overview
11/51
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.
8/8/2019 TLC Overview
12/51
Adsorbents for TLC
Silica gel
Silica gel-F (Fluorescing indicator added) Magnesium Silicate (Florisil)
Polyamides
Starch
Alumina
8/8/2019 TLC Overview
13/51
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
8/8/2019 TLC Overview
14/51
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
8/8/2019 TLC Overview
15/51
Silica Gel (3)
They are very porous
They have large surface area
These affect your separation characteristics
8/8/2019 TLC Overview
16/51
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
8/8/2019 TLC Overview
17/51
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
8/8/2019 TLC Overview
18/51
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
8/8/2019 TLC Overview
19/51
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
8/8/2019 TLC Overview
20/51
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)
8/8/2019 TLC Overview
21/51
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)
8/8/2019 TLC Overview
22/51
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
8/8/2019 TLC Overview
23/51
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
8/8/2019 TLC Overview
24/51
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.
8/8/2019 TLC Overview
25/51
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)
8/8/2019 TLC Overview
26/51
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
8/8/2019 TLC Overview
27/51
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
8/8/2019 TLC Overview
28/51
LikeDissolves Like
Polar molecules favor polar solvents and vice
versa
Polar solutes migrate faster in polar mobile phase
8/8/2019 TLC Overview
29/51
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
8/8/2019 TLC Overview
30/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
31/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
32/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
33/51
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
8/8/2019 TLC Overview
34/51
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 1:1
Pipettes or graduated cylinders can be used for these measurements
Remember: Polarity is controlled by your choice of solvents
(see Eulotropic values)
8/8/2019 TLC Overview
35/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
36/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
37/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
38/51
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
8/8/2019 TLC Overview
39/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
40/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
41/51
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 aresmall and quite handy
8/8/2019 TLC Overview
42/51
Performing the TLC Analysis:
Visualization and Interpretation (3)
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
8/8/2019 TLC Overview
43/51
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
8/8/2019 TLC Overview
44/51
Performing the TLC Analysis:
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
8/8/2019 TLC Overview
45/51
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% 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
8/8/2019 TLC Overview
46/51
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
8/8/2019 TLC Overview
47/51
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
8/8/2019 TLC Overview
48/51
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
8/8/2019 TLC Overview
49/51
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
8/8/2019 TLC Overview
50/51
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
8/8/2019 TLC Overview
51/51