Development And Validation Of Chromatographic Methods For Simultaneous Quantification Of Drugs In Bulk And In Their Formulations: HPLC And HPTLC Techniques
©2014
Textbook
109 Pages
Summary
This book details:<br>1. Development and validation of a HPTLC-densitometric method for concurrent estimation of metformin hydrochloride, pioglitazone hydrochloride and gliclazide in combined dosage form.<br>2. Development and validation of a HPTLC method for simultaneous estimation of moxifloxacin hydrochloride and dexamethasone sodium phosphate in combined pharmaceutical dosage form.<br>3. Development and validation of a RP-HPLC method for simultaneous estimation of ciprofloxacin hydrochloride and dexamethasone in combined dosage form, which is a better alternative to existing ones.<br>The developed analytical methods are simple, selective, accurate, robust, and precise with shorter analysis time for the analysis of drug/s in combined pharmaceutical dosage forms. All the developed HPTLC and HPLC methods have been validated as per ICH Q2 (R1) guideline. Developed analytical methods could boost analytical researchers to work more efficiently in the field of analytical method development and validation of Pharmaceutical dosage forms.
Excerpt
Table Of Contents
INDEX
Sr. No.
Chapter
Page No.
1 Introduction
1-26
2 Drug
Profile
27-42
3
Aims and Objectives
43-44
4 Plan
of
Work
45-47
5 Results
and
Discussion
48-79
6 Experimental
80-95
7 References
96-102
1
INTRODUCTION
Introduction to analytical chemistry
(1-4)
Analytical chemistry may be defined as the "science and art of determining the composition
of materials in terms of the elements or compounds contained". Large number of drug/s
introduced in the pharmaceutical market is increasing per year. These drugs may be either
new entities or partial structural modification/s of the existing one. Very often there is a time
delay from the date of introduction of a drug in the market to the date of its inclusion in
pharmacopoeias. This is because of the possible uncertainties in the continuous and wider
usage of these drug/s, reports of new toxicities (resulting in their withdrawal from the
market), development and introduction of better drug/s by competitor/s. Under these
conditions, standard/s and analytical procedure/s for these drugs may not be available in the
pharmacopoeia/s. It therefore becomes necessary to develop newer analytical methods for
such drugs.
Quality control is a concept, which strives to produce a perfect product by series of measures
designed to prevent and eliminate errors at different stages of production. With the growth of
pharmaceutical industry during last many years, there has been fast development in the field
of pharmaceutical analysis involving complex instrumentation. Providing simple, rapid
analytical method for complex formulation is a matter of importance.
Important reasons for the improvement of newer drug analysis methods are:
-
The drug or combinations of drug may not be official in any pharmacopoeias.
-
An appropriate analytical method for the drug may not be available in the literature
due to patent regulations.
-
Suitable analytical procedure may not be available for the drug in the form of a
formulation due to the interference caused by the formulation excipients.
-
Analytical method/s for the evaluation of the drug in biological fluids may not be
available.
-
Analytical method for a drug in combination with other drugs may not be available.
-
The existing analytical procedures may require expensive solvents and reagents. It
may also involve burdensome extraction, separation procedures and these may not be
reliable.
2
ANALYTICAL METHODS
Highly specific and sensitive analytical techniques hold the key in the designing,
development, standardization and quality control of medicinal products. They are equally
important in drug metabolism and pharmacokinetics studies, both of which are fundamental
to the evaluation of bioavailability and duration of clinical response.
Modern physical methods of analysis are very sensitive, precise and providing thorough
information from minute samples of material. They are for the most part quickly applied and
in general are readily amenable to automation. For these reasons they are now widely used in
product development, control of manufacture, quality control, as a check on stability during
storage and monitoring the use of drugs and medicines.
CLASSIFICATION OF ANALYTICAL METHODS
1) Chemical Methods
a) Titrimetric Methods; They involves
i. acid base reactions, ii. precipitations, iii. redox reactions iv. complexomeric reactions,
v. large cation reagents
b) Gravimetric Methods are
i. weighing the active ingredients after separation, ii. weighing of the residue after ignition of
the sample, iii. precipitation and weighing of the derivatives of the active ingredients
2) Instrumental methods
a) Spectroscopic techniques; There are,
i. ultraviolet and visible spectrophotometry, ii. fluorescence and phosphorescence
spectrophotometry, iii. atomic spectrometry (emission and absorption), iv. infrared
spectrophotometry, v. raman spectroscopy, vi. X-ray spectroscopy, vii. radiochemical
techniques including activation analysis, viii. nuclear magnetic resonance spectroscopy, ix.
electron spin resonance spectroscopy
3
b) Electrochemical techniques cover,
i. potentiometry (pH and ion selective electrodes), ii. conductance techniques, iii.
voltammetric techniques, iv. stripping techniques, v. coulometry, vi. electrogravimetry.
c) Chromatographic techniques; Some commonly used chromatographic techniques are
i. gas chromatography, ii. high performance liquid chromatography (HPLC), iii. high
performance thin layer chromatography (HPTLC).
d) Miscellaneous techniques
i. Thermal analysis, ii. Mass spectrometry, iii. Kinetic techniques.
e) Hyphenated techniques
i. GC - MS (gas chromatography - mass spectrophotometry), ii. ICP - MS (Inductively
coupled plasma - mass spectrophotometry), iii. GC - IR (gas chromatography - infrared
spectroscopy), iv. MS - MS (mass - mass spectroscopy)
UV-Visible Spectroscopy:
(5-6)
In electromagnetic spectrum wavelength range from 190 - 800 nm is called as Ultraviolet-
Visible (UV-VIS) region. The transitions that result in the absorption of radiation are
transitions between the electronic energy levels, in the UV - Visible spectroscopy
For an atom that absorbs UV - VIS radiation, the absorption spectrum sometimes consists of
very sharp lines, as it would be expected from a quantized process that is occurring between
two discrete energy levels. But for molecules, the UV - VIS absorption occurs over a wide
range of wavelengths that results in a broad band of absorption centered near the wavelength
of maximum absorption (
max
). The fundamental law that governs the quantitative
spectrophotometric analysis is Beer - Lambert's Law.
4
Beer's law
Beer observed a relationship holds between transmittance and the concentration of a solution,
i.e., the intensity of a beam of monochromatic light decreases exponentially with the increase
in concentration of the absorbing substance arithmetically.
Lambert's law
When a beam of light is allowed to pass through a transparent medium, the rate of decrease of
intensity with the thickness of medium is proportinal to the intensity of the light.
The combination of these two laws yields the Beer - Lambert's Law.
Mathematically it can be expressed as,
A= log (I
0
/I) = cl (for a given wavelength) ...eq. 1
Where,
A = absorbance, I
0
= intensity of light incident upon sample cell,
I = intensity of light leaving sample cell, c = molar concentration of solute,
l = length of sample cell (cm), = molar absorptivity.
The assay of single sample, which contains other absorbing substance/s, can be calculated
from the measured absorbance by using one of three principal procedures. They are, 1)
Standard absorptivity value, 2) Calibration graph and 3) Single or double point
standardization. Some of the methods used for the assay of multi component sample are, 1)
Simultaneous equation method, 2) Multi component mode, 3) Derivative spectrum method,
4) Absorbance correction method.
Chromatography
(7-18)
Earlier, chromatography used to be a separation technique, but now it can be used for
quantitation also. It involves the separation of components of a mixture which are scattered
among two phases, the mobile phase and the stationary phase. The mobile phase moves over
or through the surface of the stationary phase. As different components of the mixture have
different affinities for each phase they differ in their retention on the stationary phase, which
5
leads to their separation. The separation of components is determined by the chemical and
physical properties of the two phases and the experimental conditions (temperature and
pressure).
Modern pharmaceutical formulations are complex mixtures containing one or more
therapeutically active ingredients and a number of inert materials like disintegrant, colors,
excipients, and flavors. So as to ensure quality and stability of the final product, the
pharmaceutical analyst must be able to separate the mixtures into individual components
prior to quantitative analysis. Chromatography is the powerful technique to separate the
mixture into different components.
Chromatographic methods can be classified according to the nature of the stationary and
mobile phases. Different types of chromatography are: 1) Adsorption, 2) Partition, 3) Ion
exchange, 4) Size exclusion or gel permeation. The modern instrumental techniques of GLC
and HPLC provide excellent separation and allow accurate assay of very low concentrations
of wide variety of substances in complex mixtures.
High Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) is used in almost all sectors. Most of the
drugs in dosage forms can be analyzed by this technique because of several advantages like
accuracy, precision, specificity, and ease of automation.
There are different modes of separation in HPLC. They are normal phase and reversed phase,
reverse phase ion pair, affinity chromatography and size exclusion chromatography (gel
permeation and gel filtration).
In the normal phase, the mobile phase is nonpolar and the stationary phase is polar in
nature. In this method, nonpolar compound/s travel quicker and are eluted first, because of
the lower attraction between the nonpolar compound/s and the stationary phase. Polar
compound/s are retained for longer period of times because of their higher affinity with the
stationary phase. These compounds, therefore take more time to elute. Hence, normal phase
separation is not commonly used for pharmaceutical applications because most of the drug
molecules are polar in nature and hence take longer time to elute. In reversed phase, the
stationary phase is nonpolar and the mobile phase is polar.
6
An aqueous mobile phase allows the use of secondary solute chemical equilibrium (such as
ionization control, ion pairing, ion suppression and complexation) to control retention and
selectivity. The polar compounds get eluted first in this mode and nonpolar compounds are
retained for longer time of period. As nearly all the drug/s and pharmaceuticals are polar with
varying degree in nature, they are not retained for longer times and thus elute more rapidly.
The different HPLC columns used are C4, C8, octa decyl silane (C18 or ODS) etc.
In ion exchange chromatography, the stationary phase contains ionic groups like NR
3+
or
SO
3-
, which interact with the oppositely charged ionic groups of the sample molecules which
is suitable for the separation of charged molecules only. Altering the salt concentration and
pH can change the retention.
Ion pair chromatography may be used for the separation of ionic compounds and this
method can also be a substitute for ion exchange chromatography. In reversed phase ion pair
chromatography or soap chromatography, strong basic and acidic compound/s may be
separated by reversed phase mode by forming ion pairs (columbic association species formed
between two ions of opposite electric charge) with appropriate counter ions.
Affinity chromatography uses highly specific biochemical interactions for separation
process. The stationary phase contains specific groups of molecules which can adsorb the
sample if certain steric and charge related conditions are satisfied. This technique can be used
to isolate enzymes, proteins, antibodies from complex mixtures.
Size exclusion chromatography separates molecules according to their molecular mass.
Molecules with highest molecular mass are eluted first and the smallest molecular mass in the
last. This technique is usually used when a mixture contains compounds with a molecular
mass difference of at least 10%. This mode can be further subdivided into gel permeation
chromatography (with organic solvents) and gel filtration chromatography (with aqueous
solvents).
Column packing
The packing used in modern HPLC consists of small, rigid particles having a narrow particle
size distribution. The different types of column packing in HPLC are
7
1. Porous, polymeric beds
Porous, polymeric beds based on styrene divinyl benzene co-polymers. They are used in ion
exchange and size exclusion chromatography. For analytical purpose these are replaced by
silica based packing which are more efficient.
2. Porous layer beds
Consisting of a thin shell (1-3 m) of silica or modified silica on a spherical inert core (e.g.
glass). After the development of totally porous micro particulate packing, these have not been
used in HPLC.
3. Totally porous silica particles (dia. < 10 m)
These packing have been widely used for analytical HPLC in recent years. Particles of
diameter > 20 m are usually dry packed, while particles of diameter < 20 m are slurry
packed in which particles are suspended in a suitable solvent and the slurry so obtained is
driven into the column under pressure.
Detectors
The function of the detector in HPLC is to monitor the mobile phase as it merges from the
column. Usually, detectors are of two types:
1. Bulk property detectors
It compares overall changes in a physical property of the mobile phase with or without an
eluting solute. e.g. dielectric constant or density, refractive index.
2. Solute property detectors
It responds to a physical property of the solute. e.g. Fluorescence, UV absorbance or
diffusion current. These detectors are about 1000 times extra sensitive than the bulk property
detectors.
8
Quantitative analysis in HPLC
Three methods are generally used for quantitative analysis. They are as follows,
1. External standard method
The external standard method involves the use of a single standard. The peak area or the
height of the sample and the standard used are compared.
2. Internal standard method
A widely used technique of quantitation involves the addition of an internal standard to
compensate for various analytical errors. In this method, a known drug of a fixed
concentration is added to the known amount of samples to give separate peaks in the
chromatogram to compensate for the loss of the compounds of interest during pretreatment of
the sample. Loss of the component of interest will be accompanied by the loss of an
equivalent fraction of the internal standard. The correctness of this method clearly depends on
the structural equivalence of the compounds of interest and the internal standard. The
necessities for an internal standard are:
-
It must give a completely resolved peak with no interferences.
-
It should elute close to the compound of interest.
-
It must behave equivalent to the compound of interest for analysis like
pretreatments, derivative formations etc.
-
It should be added at a concentration that will produce a peak area or peak height
ratio of about unity with the compound.
-
It should not be present in the original sample.
-
It must be stable, nonreactive with column packing, the mobile phase, sample
components, and
-
It is desirable that this compound is commercially obtainable in high purity.
The internal standard should be added to the sample prior to sample preparation procedure
and homogenized with it. To be able to recalculate the sample component concentration in
the original sample, the response factor should be demonstrated first. The response factor
(RF) is the ratio of peak areas of sample component (Ax) and the internal standard (AISTD)
9
obtained by injecting the same quantity on molar basis. It can be calculated by using the
formula,
RF = Ax / AISTD ...eq. 2
On the basis of the response factor and strength of the internal standard (NISTD), the amount
of the analyte in the original sample can be calculated using the formula,
X =Ax × N / RF × AISTD ...eq. 3
The calculations described above can be used after proving the linearity of the calibration
curve for the internal standard and the analytical reference standard of the compound of
concern. While more than one component is to be analyzed from the sample, the response
factor of each component should be determined. One can also use the slope of the calibration
curve based on standard that contain known concentrations of the compound of interest.
When more than one component is to be analyzed from the sample, the response factor of
each component should be determined in the calculations using similar formula.
3. Standard addition method
In this technique, a known quantity of the standard compound is added to the sample solution
to be determined. Standard addition technique is suitable if sufficient amount of the sample is
available and is more realistic in the sense that it allows calibration in the presence of
excipients or other components. As gradient elution can be a source of additional error in
quantitative study. It is also essential to maintain the flow rate and the mobile phase
composition steady. The sample must be dissolved in the mobile phase. If the solvent used in
preparing the sample solution and the mobile phase are not the same, the study can become
less precise.
System suitability Parameters:
To ensure that the data obtained from HPLC system and procedure followed is of acceptable
quality, system suitability testing should be performed. System suitability testing is an
integral part of chrmatographic methods. A few commonly used system suitability parameters
and what they mean to analysis is given below. System suitability tests must be performed on
a regular basis.
10
i) Resolution (R
S
): -
It is a measure of quality of separation of adjacent bands in a chromatogram; obviously
overlapping bands have small Rs values. Resolution is calculated from the retention time and
the width of two adjacent peaks.
Ideally R
S
should be greater than 1.5
2 (t
2
- t
1
)
w
1
+ w
2
Where t
1
and t
2
are the retention time of the first and second eluting adjacent bands, where
W
1
and W
2
are their respective baseline widths. Reliability of calculation is poor if Rs is <
2.0.
ii) Capacity factor (k): -
It is the measure of the position of a sample peak in the chromatogram, being specific for a
given compound. This is a parameter that specifies the extent of the retention of substances to
be separated. It depends on the mobile phase, quality of column packing, stationary phase and
temperature.
t
1
- t
0
t
0
t
2
- t
0
t
0
Where,
t
0
= Dead time of column
t
1,
t
2
= Retention times of 1
st
and 2
nd
eluting components
...eq. 4
R
S
=
...eq. 5
k
1
=
...eq. 6
k
2
=
11
iii) Selectivity factor:
The selectivity of the chromatographic system is a measure of the difference in retention time
between two given peak. The selectivity factor ( ) of a column for the two species A and B is
defined as;
= K
B /
K
A
...eq. 7
Where K
B
is distribution constant for more strongly retained species B, and K
A
is distribution
constant for less strongly or more rapidly eluted species A. Alpha is always greater than
unity.
iv) Number of theoretical plates (N): -
The column efficiency can be expressed as the plate number (N) and Height equivalent to
theoretical plate [HETP]
t
R
2
W
N = number of theoretical plates
t
R
= retention time of the component (min)
w = width at the base of the peak,
v) Height equivalent to theoretical plates (HETP): -
L
N
Where,
H = Height equivalent to theoretical plates (HETP),
L = Length of column,
N = Number of theoretical plates.
...eq. 8
N=
...eq. 9
HETP = H =
12
vi) Tailing factor (T): -
The tailing factor (a measure of peak symmetry), is unity for perfectly symmetrical peaks and
its value increases as tailing becomes more pronounced.
W
0.05
2f
Where,
f = distance between maxima of two peaks
W
0.05
= width of peak at 5 % height. Ideally T value should be
d 2.
Design of Separation Method
To develop a method for analyzing single or multiple components of a formulation one
should know the nature of sample present like, molecular weight, polarity, ionic character and
solubility. Development of a method involves considerable trial and error procedures.
Generally, one starts with reversed phase chromatography which involves non polar
stationary phase, when the compounds are hydrophilic in nature and are water soluble.
The organic solvent concentration required for the mobile phase can be estimated by gradient
elution technique. In case of aqueous sample mixtures, the best way to start is with gradient
reversed phase chromatography. Gradient can be started with 5 - 10 % organic solvent in the
mobile phase and the organic solvent concentration can be increased up to 100 % within 30 -
45 min. Separation can then be optimized by changing the initial mobile phase composition
and the slope of the gradient according to the chromatogram obtained from the preliminary
chromatographic run. Initial mobile phase composition can be estimated on the basis of
where the compounds of interest eluted, namely at what mobile phase composition. Changing
the polarity of mobile phase can alter elution of drug/s. Elution strength of a mobile phase
depends upon its polarity. Ionic samples (acidic/ basic) can be separated, if they are present in
unionized form. Dissociation of ionic samples may be suppressed by the proper selection of
pH. The pH of the mobile phase has to be selected in such a way that the compounds do not
ionize. If the retention times are too short, the organic phase concentration needs to be
...eq. 10
T =
13
decreased. If the retention time is very high, organic phase concentration needs to be
increased.
Selection of detection wavelength is an important activity to get good analytical results. To
select a wavelength of detection one should have the knowledge of UV spectrum of each
component in the sample. UV spectra can be measured for standards prior to method
development. It is not always necessary to use
max
for the detection. For simultaneous
method development, a wavelength where all the components of the sample have
considerable absorption should be selected considering the ratio of components in the
formulation. The addition of peak modifiers to the mobile phase can affect the separation of
ionic samples. For examples, the retention of the basic and acidic compounds can be
influenced by the addition of small amounts of triethylamine and acetic acid (a peak
modifier), respectively. This can lead to valuable changes in selectivity. Tailing or fronting
indicates that the mobile phase is not totally compatible with the solutes. Ion - pair
chromatography can be used, when the peak shape does not improve by lower (2 - 3) or
higher (8 - 9) pH. For basic compounds, anionic ion - pair molecules at lower pH and acidic
compounds, cationic ion pair molecules at higher pH can be used. In case of amphoteric
solutes or a mixture of acidic and basic compounds, ion-pair chromatography is the technique
of choice. The low solubility of the sample in the mobile phase can also results in to bad peak
shapes. It is always suitable to use the same solvents for the preparation of sample solution as
the mobile phase to avoid precipitation of the compounds in the system. Optimization can be
started only after a reasonable chromatogram (chromatogram with more or less symmetrical
peaks) has been obtained. By small alter of the mobile phase composition, the peak position
can be predicted within the range of investigated alterations. An optimized chromatogram is
the one in which all the peaks are symmetrical and are well resolved in less run time. The
peak resolution can be increased by using a more efficient column (column with higher
theoretical plate number, N) which can be obtained by means of a column of smaller particle
size, or a longer length. However, these factors, will enhance the analysis time.
High Performance Thin Layer Chromatography (HPTLC)
HPTLC is a simple separation technique in which different samples are applied to the
stationary phase before it comes in contact with the mobile phase resulting in sample
migration. After development the mobile phase is removed by evaporation and detection is
performed on the stationary phase. The record of the detector response is plotted against the
14
separation distance is called a densitogram. Availability of different stationary phases is an
important difference between TLC and column chromatography. Each run needs use of new
stationary phase which eliminates the cross contamination from previous samples. Only the
sample components that are eluted out of the column can be detected in the column
chromatography and the components that remain on the column may be easily overlooked but
in TLC components cannot be usually overlooked. As the mobile phase is evaporated before
the detection process, it does not interfere with the measurement of components of the
mixture. In classical TLC, mobile phase moves through the stationary phase by capillary
forces. There are many modifications to the classical TLC approach in which flow of mobile
phase is forced through the layer which are collectively called as forced flow methods. Some
of the forced flow methods are electro-planar chromatography (EPC), over pressure layer
chromatography or optimum performance laminar chromatography (OPLC), rotation planar
chromatography (RPC) etc. Capillary forces are stronger in the narrow inter-particle
channels, leading to more rapid advancement of the mobile phase. During the
chromatographic process a solvent gradient in the mobile phase is produced as the solvent
front migrates through the adsorbent layer. This is particularly true for mixed mobile phases
where more polar component is more selectively adsorbed. If vapor and mobile phase are not
in equilibrium, evaporation causes loss of mobile phase from the plate surface.
TLC provides for separations in the milligram to picogram range. Separated substances that
are alternately identified by TLC can be isolated for further characterization by other
techniques, such as gas chromatography (GC), high performance liquid chromatography
(HPLC), visible, ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR), mass
spectrometry (MS), and electrophoresis. Eluted substances can also be quantified by
procedure such as these, but in situ densitometry is the most convenient, accurate, and precise
approach for quantitative TLC. The fundamental parameter used to characterize the position
of a spot in a TLC chromatogram is the Retardation factor or R
f
value.
The Retardation Factor (R
f
)
It is the quotient of the distance of the substance zone from the sample origin to the front of
the mobile phase (Z
f
).
15
Z
s
Z
f
- Z
o
Where, Z
s
= distance of the substance zone from the sample origin (mm).
Z
f
= solvent front migration distance (mm).
Z
o
= distance between immersion line and sample origin (mm).
Fig. No.1 Calculation of R
f
Value
By definition, the R
f
value cannot exceed 1.0. To avoid the decimal point, the R
f
value is
sometimes multiplied by 100 and then described as the hR
f
value. Systematic error in the
measurement of the R
f
value arises from the difficulty in locating the precise position of the
solvent front. If saturation of the development of the chamber is not done, adsorbent layer,
mobile phase and vapor phase will be in equilibrium, then the condensation of vapor phase or
the evaporation of mobile phase in the region of the solvent front will give erroneous R
f
value.
The ultimate chromatographic performance of a TLC plate, and therefore resolution, is
dependent on certain parameters like the velocity constant of mobile phase, diffusion
coefficient of the substance in mobile phase, mean particle size and particle size distribution
of the stationary phase. The mobile phase velocity is determined by particle size while
chromatographic efficiency is dependent upon the coarser particles and the performance is
improved by using particles of narrow size distribution. However, spot broadening in HPTLC
R
f
=
...eq. 11
16
is controlled by molecular diffusion. Performance of TLC can be evaluated in terms of the
number of theoretical plates (N), height equivalent to theoretical plate (HETP), and
separation number (SN).
In column chromatographic techniques, all substances travel same migration distance (the
length of column) but have different diffusion time (retention time on the column). This is
opposite to TLC where all substances have same diffusion time (the plate is developed for the
fixed time) but migration distance varies. The chromatographic measures of performance in
TLC (N, HETP, and SN) are all correlated to the migration distance of substance.
Advantages of HPTLC
They are; 1) short development time, 2) wide choice of stationary phases, 3) early recovery of
separated components, 4) superior separation effects, 5) easy visualization of separated
compounds.
Steps involved in HPTLC analysis
i)
Sample preparation: -
For normal phase chromatography using silica gel / alumina precoated plates, solvent
generally should be nonpolar and of volatile type. For reverse phase chromatography
usually polar solvents are used.
ii)
Selection of chromatographic layer: -
There are at least 25 types of sorbents available for TLC. Silica gel or aluminium oxide
is useful in many applications. They also can be split into different types depending on
the pore size, particle size and pH. Selection of layer depends on the nature of material to
be separated like polarity, solubility, ionizability, molecular weight, shape and size.
These properties are also important for selecting the solvents for preparation of sample
and development. Types of sorbents are given below,
17
Silica based
Silica gel bonded phases Silica gel
Reverse Amino bonded Cyano bonded Diol bonded Chiral bonded
Phases phases phases phases
phases
Non-silica based
Cellulose bonded phases Cellulose
PEI cellulose
Acetylated Carbxymethyl(CM) and
(polyethyleneimine) cellulose diethylaminoethyl celloulose
18
Table No.1
Choice of optimum TLC/HPTLC sorbents for compounds and compound classes
(Peter E Wall et al., Thin Layer Chromatography A modern practical approach, © The
Royal Society of Chemistry 2005.pp. 7)
Sorbents Compounds
separated
Silica gel
All classes of compounds
Aluminium oxide
Basic compounds (alkaloids, amines etc.), terpenes, steroids,
aromatic and aliphatic hydrocarbons.
Cellulose
Food dyes, amino acids and derivatives, carbohydrates.
Kieselguhr Aflatoxins,
herbicides, carbohydrates, tetracyclines.
Polyamides Phenols,
flavonoids,
nitro
compounds.
Amino bonded silica
gel
Particularly good for phenols, carboxylic acids,
carbohydrates, sulfonic acids, nucleotides, nucleosides.
Cyano bonded silica
gel
Particularly good for pesticides, steroids, preservatives.
Diol baonded silica gel Particularly good for steroids, hormones.
Reversed phase silica
gel
Improves separation for many classes of compounds
Chiral modified silica
gel
Enantiomers of amino acids, halogenated, N-alkyl, and -
methyl amino acids, simple peptides, -hydroxy carboxylic
acids (catecholamines).
Silica gel impregnated
with silver nitrate
Lipids, including variations in unsaturation and geometric
isomers.
19
iii)
Plates: -
Standard size plates for HPTLC are manufactured by various companies which are most
satisfactory. Generally plates of 20 X 20 cm, 10 X 10 cm or 5 X 7.5 cm size having 100 -
250 adsorbent thickness are used for quantitative analysis. Silica gel 60F
254
having a
pore size 6 with fluorescent indicator as a coat material is widely used. The basic
difference in TLC and HPTLC plate is particle size of coated material which is 5 - 20 m
for TLC and 4 - 8 m for HPTLC.
iv)
Pre-washing: -
Plates need to be prewashed to remove water vapors or other volatile impurities, which
might get trapped in the plates. These give dirty zones and spots on the plates. To avoid
this, plates are cleaned by using methanol as solvent by ascending or descending or by
dipping mode.
v) Conditioning: -
The prewashed plates exposed to humidity and surroundings are needed to be activated
by placing them in an oven at 110° C for 15 to 20 minutes. This process is known as
conditioning. This allows the active centers of coating materials attenuated for better
separation of sample material.
vi) Sample application: -
It is most important step for obtaining good resolution and results. Application of 1.0 - 5
L is most satisfactory, for HPTLC, application of the sample and standard as a band
gives better separation, equal R
f
values and less spot broadening. This sample application
is carried out by Linomat type applicator on the plates which gives uniform, accurate
results. In Linomat applicator nitrogen gas is used for the sample application. Flow of
nitrogen is adjusted according to vehicle used for sample preparation.
vii) Pre conditioning (Chamber saturation): -
This has profound influence on the effective separation of sample. For low polarity
mobile phase there is no need of saturation, however, saturation is desirable in case of
highly polar mobile phases. Partial saturation is recommended for mobile phase
20
composition leading to phase separation. For reverse phase chromatography it is essential
to saturate the chamber with methanol or polar solvent.
viii) Mobile phase: -
The selection of appropriate mobile phase is based on chemical properties of solute and
solvent, solubility of analyte, absorbent layer etc.
ix) Chromatographic development: -
Various forms of chromatographic development like ascending, descending, horizontal,
continuous, and gradient can be tried. For HPTLC plates, migration distance of 5- 6 cm
is sufficient. After development, plates are removed from the chamber and dried to
remove traces of mobile phase.
There are some special development techniques in TLC, they include
- Continuous development
- Multiple development
- Stepwise development
1)
Two dimensional separation
2)
Three dimensional separation
3)
Wedged tip technique
Common problems encountered during chromatographic development are as follows;
(a)
Tailing: -
This may occur due to the presence of traces of impurities or due to presence of more than
one ionic species of substances being chromatographed. This can be reduced by buffering the
mobile phase with acidic (1 - 2 % acetic acid) or basic (ammonia) solution. It keeps the
materials to be separated in non ionic forms.
21
(b)
Diffusion: -
Diffusion may arise due to non uniformity of mobile phase, longitudinal diffusion between
stationary phase and mobile phase or due to non equilibrium of stationary phase.
x) Detection of spots: -
Immediately after the development step is completed, the plates are removed from the
chamber and dried to remove traces of mobile phase. Generally detection can be done by
derivatization with iodine vapors or using sulfuric acid. Alternatively, detection can be done
by visual inspection at 254 nm or 366 nm in UV cabinet.
xi) Scanning and Documentation: -
Nowadays HPTLC equipments are supplied with computer and data recording and scanning
devices. The developed HPTLC plates are scanned at selected wavelengths (UV) by the
instrument and the detected spots are seen on computer in the form of peaks. The scanner
converts band into peaks and peak height or area is related to the concentration of the
substance on the spot.
Fig. No. 2. Representation of bands in the form of peaks by scanner
22
Factors affecting HPTLC separation are as follows: -
1) Type of stationary phase, 2) mobile phase, 3) layer thickness and binders in the layers, 4)
solvent purity, 5) size of the developing chamber, 6) saturation of the chamber (pre-
equilibrium), 7) sample volume to be spotted, 8) relative humidity, 9) temperature (R
f
values
usually increase with rise in temperature), 10) flow rate of solvent, 11) development distance.
Analytical Method Validation:
19
ICH guidelines on analytical method validation Q
2
(R
1
) describe the characteristics for
consideration during the validation of the analytical procedures. The objective of validation
of an analytical procedure is to demonstrate that it is suitable for its intended purpose.
Analytical procedure
The analytical procedure refers to the way of performing the analysis. It must describe in detail
the steps essential to perform each analytical test. This may comprise but is not limited to: the
sample, the reference standard, use of the apparatus, reagents preparations, use of the formulae
for the calculation, generation of the calibration curve etc.
Typical validation characteristics of analytical method validation include,
1)Accuracy, 2)Precision (Repeatability, Intermediate Precision), 3) Specificity, 4) Detection
Limit, 5) Quantitation Limit 6) Linearity 7) Range, 8) Robustness 9) Ruggedness
1) Accuracy
The accuracy of an analytical procedure expresses the closeness of an agreement between the
value, which is accepted either as a conventional true value/accepted reference value and the
found value. This is sometimes termed as trueness. It is often expressed as % recovery by
analyzing known added amounts of analyte. An example of an accuracy criteria for an assay
method is that the mean recovery will be 100 ± 2 % at each concentration over the range of 80
- 120 % of the target concentration.
2) Precision
The precision of analytical procedure expresses closeness of agreement (degree of scatter)
between a series of measurements obtained from multiple sampling of the same homogenous
Details
- Pages
- Type of Edition
- Erstausgabe
- Publication Year
- 2014
- ISBN (eBook)
- 9783954898077
- ISBN (Softcover)
- 9783954893072
- File size
- 2.4 MB
- Language
- English
- Publication date
- 2015 (August)
- Keywords
- development validation chromatographic methods simultaneous quantification drugs bulk their formulations hplc hptlc techniques
