Pharmaceutical Instrumental
Analysis
PHC 427
Dr. haya Al-johar
Chief of Research and Seized Department
Saudi Food & Drug Authority
E-mail : hijohar2@hotmail.com
LECTURES’ OUTLINE
High performance liquid chromatography (HPLC)
Analytical features of HPLC
External and internal standard methods
Stability-indicating methods of assay.
Chiral separation of pharmaceutical compounds
Chiral separation of pharmaceutical compound
Separation and quantification of related Substances
Gas Chromatographic
The thermodynamic of gas chromatography.
Instrumentation of gas chromatography.
 Application of gas chromatography.
Capillary electrophoresis
Principles and instrumentation
Choice of optimum conditions for resolution.
 Modes of electrophoretic separation
Applications of capillary electrophoresis
Atomic absorption and emission spectrophotometry
Instrumentation of atomic absorption
Quantitative analysis by of atomic absorption
 Principles of atomic emission
 Instrumentation of atomic emission.
 Applications of atomic emission
High Performance Liquid
Chromatography
(HPLC)
Identification:
It is apart of chromatography techniques
(physical separation) which is a general term
applied to a wide variety of separation methods.
based on the sample partitioning between a moving
(mobile) phase, which can be a gas, liquid, or
supercritical fluid, and a stationary phase, which
may be either a liquid or a solid.
History:
The discovery of chromatography is generally
credited to Tswett, who in 1906 described his
work on using a chalk column to separate
pigments in green leaves.
1940 paper chromatography .
1950 gas and thin-layer chromatography.
1960 various gel or size-exclusion methods
Classification of chromatographic methods
Gas
Adsorption
Chromatography
Competition between a solid
adsorbent and the mobile
phase
St. solid
mo. liquid
column
Liquid
Planer layer
Partition
Chromatography
Competition between a
liquid stationary phase and
the mobile phase
St. liquid
Mo. liquid
Ion Exchange
Chromatography
Competition between an ion
exchange resin stationary
phase and liquid mobile
phase
Permeation
Chromatography
Competition between a
polymer matrix and liquid
mobile phase
column
Gas Solid Chrom. (SSC or
GC)
Liquid Column Chrom. (LC)
High Performance Liquid
Chromatography (HPLC)
Thin Layer Chrom. (TLC)
Paper Chrom. (PC)
Gas
column
Gas Solid Chrom. (GLC)
Liquid
column
Liquid Column Chrom. (LC)
High Performance Liquid
Chrmatography (HPLC)
column
Liquid
Liquid
column
Ion Exchange Chrom. (IEC)
Gel Permeation Chrom.
(GPC)
applications of hplc:
high-performance liquid chromatography
(HPLC) is an analytical technique that is widely
used for:
separation,
Identification (Qualitative analysis)
Determination (Quantitative analysis)
of the chemical component in a mixture and
Preparation of interest components.
It is the major and integral analytical tool
applied in all stages of drug discovery,
development, and production of (API).
HPLC is required when:
•a mixture is too complex for direct analytical
method e.g. spectroscopy.
•the materials to be analyzed are very similar
e.g. isomer.
•it is necessary to prepare highly purified
material.
•a measurement of the amount of a particular
material is needed.
In chromatographic process, there are 3 steps:
•Injection.
•Separation.
•Elution.
Injection
Separation
Elution
Separation Mechanism
Mobile phase (solvent)
↓ ↓ ↓ ↓
A
B
B
C
C
A
Column
time
Packing
material
C>B>A
Separation is determined by column (packing
material) and mobile phase (solvent)
The migration of the sample component
through the stationary phase is a result of
two forces:
movement driven by the mobile phase.
retardation resulting from the stationary
phase.
Thus, the sample molecules are held by the
stationary phase and transported by the
mobile phase.
Results obtained by HPLC
C
A
B
Chromatogram containing three peaks
Qualitative analysis (identification) and
Quantitative analysis (determination)
Can be performed using the information contained in the
chromatogram
Identification
C
What is component A?
A
B
Sample
Caffeine
Component (A ) elutes the same time as a caffeine peak.
Component (A) is identified as caffeine.
Determination
What is the concentration
of component A?
C
A
B
Caffeine (1mg/ml)
5ul injection (5ug)
Peak area (or height) is proportional to the concentration
(or amount) of the component.
The concentration of component A (caffeine) is determined
by comparing the peak area with that of the standard caffeine
peak.
HPLC Instrumentation
Data
processor
Column
oven
Pump
Pump
Mobile phase
reservoir
Injector
Auto
sampler
Column
Detector
Reagent
pump
Fraction
collector
waste
HPLC Apparatus
Mobile phase reservoirs
A modern HPLC apparatus is equipped with one or
more glass reservoirs each of which contain 500
ml or more of the mobile phase.
Often the reservoirs contain a filtration system
for filtering dust and particulate matters from
the solvent to prevent these particles from
damaging the pumps or injection valves or
blocking the column.
HPLC Apparatus
The mobile phase usually produce bubbles in the column
and thereby can cause “band spreading” and interfere
with the performance of the detector. Therefore, the
solvent used must be “degassed” to overcome the
dissolved gases.
Degassers may consist of a vacuum system, ultrasonic
vibration, a system of heating and stirring with reflux
condenser, helium or system of sparging.
An elution of single solvent system (e.g. 50% water
+ 50 methanol) of constant composition is termed
“isocratic”.
In “gradient elution” two or more solvent system
that differ in polarity are employed, e.g. methanl
(10%, 20% and 30%), and water (90%, 80% and
70%). o
Isocratic versus Gradient Elution
Isocratic elution has a constant mobile phase composition
Gradient elution has a varying mobile phase composition
HPLC Apparatus
Pumping System
The function of the pump in HPLC is to pass the
mobile phase through the stationary phase at high
pressure and at a controlled flow rate. A pump
capable of pumping solvent up to 4000 psi and at
flows of up to 10 ml/min the Action of the Pump is
most critical, since it must not mixed up the sample
being analyzed with the solvent causing loss of
resolution. The pump must be made of material,
such as stainless steel or teflon; that resist the
chemical reaction with the mobile phase.
HPLC Apparatus
Injector System
The Function of the injector is place the
sample into the high pressure flow as narrow
volume so the sample inters the column as
homogenous.
There are two main types of injectors:
1) Fixed loop injector.
2) Variable volume injector.
The fixed loop has the advantage of high
precision of injector volume. But the loop must be
overfilled by several times its volume, that is, a 10
l loop requires 30 – 50 l to ensure that it is
filled completely with the sample.
HPLC Apparatus
The variable volume injector, as its name implies,
can accommodate volumes from 1 l to 2ml. this has
an advantage in that for limited sample sizes and for
large preparative work, the same injector may be
used without mechanical changing of the loops.
Columns
Liquid chromatographic columns are constructed
from smooth-bore stainless tubing or heavy walled
glass tubing. the majority of columns range in length
from 10 cm—30 cm, the inside diameter is often from
4 mm to 10 mm ; the common particle size of packing
are 3, 5, and 10 m the common column in use is one
that is 25 cm in length, 4.6 mm in inside diameter and
packed with 5 m particles.
HPLC Apparatus
Detectors
The characteristics of an ideal detector for
HPLC include the followings:
a) should have a high sensitivity.
b) should respond universally with all solutes.
c) should have a linear response over several
order of concentrations.
d) should be insensitive to temp changes & the
mobile phase velocity change.
e) should be reliable and convenient to use.
Unfortunately, no single detector satisfies all
these criteria.
HPLC Apparatus
The detector used in HPLC depends on the nature
of the sample.
The most widely used detectors in HPLC are:
UV detector
HPLC Apparatus
Diode Array Detector (DAD)
HPLC Apparatus
Evaporative Light Scattering Detector (ELSD)
HPLC Apparatus
Electrochemical detector
HPLC Apparatus
Refractive Index detector
HPLC Apparatus
Flourescence detector
HPLC Apparatus
HPLC recorder
A data capture system, which may be a computing
integrator or a PC with software suitable for
processing chromatographic data
modes in HPLC
LC mode
Normal phase
Reversed phase
Packing materials
Silica gel
a Silica-C18(ODS)
Mobile phase
n-Hexane/IPE
Adsorption
MeOH/Water
Hydrophobic
Size exclusion
Porous polymer
THF
Ion exchange
Ion exchange gel
Buffer sol.
Packings with
ligand
Buffer sol.
Affinity
Interaction
Gelpermeation
Ion exchange
Affinity
HPLC Modes
1- Partition Chromatography (PC)
the stationary phase is a liquid coated or linked to a
solid support
retention is due to the partitioning of the solute
between the two liquid phases (relative solubility)
separation is based mainly on differences between the
solubilities of the components in the mobile and
stationary phases (liquid liquid chromatography)
Cm
Cs
HPLC Modes
Two types of partition chromatography are
encountered,
namely,
normal
phase
partition
chromatography
(NPPC)
and
reversed
phase
partitioning chromatography (RPPC).
In normal phase the stationary phase is normal
“polar” (such as triethylglycol or water) & the mobile
phase is non polar (such as hexan or diethylether).
HPLC Modes
In reversed phase chromatography, the
stationary phase is non polar (such as C18
phase, C8 phase) and the mobile phase is
relatively polar (such as water, methanol)
Reversed methods are the most commonly
used to prepare bonded phase from silica
involves the reaction of the silica with a
substituent such as dimethyl chloro silane.
HPLC Modes
R group may be hydrocarbon such as C18 or
C8 or a hydrocarbon with a polar group such
as CN or NH2.
e.g. to prepare C18 phase.
In normal phase chromatography, the least polar
elute first. In contrast in the reversed phase the
most polar component elute first. “Like Attracts Like,
and Opposites are Not Attracted”
“Like Attracts Like, and Opposites are Not
Attracted”
Reversed-Phase
Highly
polar
CH3
CH3
Highly
polar
CH3
CH3
CH3
CH3
CH3
CH3
Moderately
polar
Non
polar
Polar mobile phase
Moderately
polar
No-Polar stationary phase
Non
polar
Non-polar mobile phase
Normal-Phase
HPLC Modes
2.Adsorption Chromatography (AC)
In adsorption chromatography, the analyte
species are adsorbed on either the surface of
polar solid stationary phase such as silica,
alumina, porous glass, when the mobile phase is
relatively non polar such as hexane(NPAC) or
the surface of non polar solid stationary phase
such as polymer beads when the mobile phase is
polar, such as water or acetonitrile (RPAC).
In adsorption chromatography, the only variable
that affect the partition coefficient of
analytes is the composition of the mobile phase,
in contrast with partition chromatography when
the polarity of the stationary phase can also be
varied.
Solvent
Structure
Cyclohexane
Dielectric Constant (25°C)
1.89
Hexane
CH3(CH2)4CH3
2.02
Carbon tetrachloride
CCl4
2.24
Benzene
2.28
Diethyl ether
CH3CH2OCH2CH3
4.34
Chloroform
CH3Cl
4.87
Ethyl acetate
Tetrahydrofuran (THF)
O
H3C C OCH2CH3
O
6.02
7.52
CHCl2
9.14
Acetone
O
H3C C CH3
20.7
Ethanol
CH3CH2OH
24.3
Methanol
CH3OH
33.6
Acetonitrile
CH3CN
36.6
Dichloromethane
Dimethylformamide (DMF)
Water
O
H C N(H3)2
H2O
38.3
78.4
The effect of pH on the HPLC retention time
An additional factor which can be used to control the
solvent strength of the mobile phase is pH.
Control of he rate of elution via the pH of the mobile
phase is only applicable to compounds in which the
degree of ionization dependent on pH but this covers a
majority of commonly used drugs.
The pH of the mobile phase can only be within the
range of 2-8.5 pH units because of the tendency for
extremes of pH to dissolve silica gel and break the
bonds between silane-coating agents and the silica gel
support.
The effect of pH on the HPLC retention time
The effects of pH on retention time are as yet not fully
understood.
The greatest effects of alteration of pH in the mobile
phase are observed within one pH unit either side of the
pKa value of the drug, i.e: where the partition
coefficient of the partially ionized drug varies between
90% and 10% of the partition coefficient of the unionized drug.
HPLC Modes
3-Size exclusion chromatography (SEC)
the stationary phase is a porous material having
controlled pore size
separation is based mainly on exclusion effects,
such as differences in molecular size and/or
shape
the terms Gel Filtration and Gel-Permeation
Chromatography
(GPC) were used earlier to describe this process
HPLC Modes
4-Ion exchange chromatography (IEC)
the stationary phase has ionically charged
groups at the surface
the retention is due to the attractive
interactions between ionic solutes and the
opposite charged stationary phase
separation is based mainly on differences in
the ion exchange affinities of the sample
components
this technique is now often referred to as Ion
Chromatography (IC)
Theoretical Consideration
Retention Time (tR)
The retention time (tR) is the time between injection
of a sample and the appearance of a solute peak at
the detection of a chromatographic column.
The retention volume (VR) is the volume of mobile
phase required to elute a component from the column.
VR = F  t R
where F is the volume flow rate of the mobile phase.
Theoretical Consideration
Capacity Factor (KA)
The capacity factor is a measure of the
position of a sample peak in the chromatogram. It is
specific for a given substance. KA depdnds on the
stationary phase, the mobile phase, the temperature
t R  to
KA 
to
or
vt  vo
KA 
vo
where,
t0 = the time taken for unretained molecule to pass through the void volume
tR = the time taken for the analyte 1 to pass through the column
vo = the void volume of the column
vt = retention volume of the analyte.
A large capacity factor favors good separation and leads
to increase the elution time (2-10 min).
Theoretical Consideration
Theoretical Consideration
The relative retention ()
The relative retention, also known as separation
factor, is the ratio between two capacity
factors. It is a measure of the column’s ability
and mobile phase to discriminate two compounds:
K A2

K A1
If the  = 1, no separation has occurred. The larger
the  value, the easier the HPLC separation is to
achieve.
.
An  value
of the 1.1—10 is typically desired.
Theoretical Consideration
Resolution (Rs)
Resolution (Rs) describes the degree of separation
between two adjacent bands
V V 
Rs  2 R 2 R1 
 W 1  W2 
The greater the value for Rs, the better the
separation of the two compounds. When Rs = 1, the
two bonds one reasonably well separated; that is
only 2% of one band overlaps the other.
Theoretical Consideration
Number of theoretical plates (N)
The separation efficiency of a column can be
expressed in terms of number of theoretical
plates in the column (N).
 tR 

N  5.54
 W1/ 2 
2
or
 tR 
N  16 
W 
2
Theoretical Consideration
For high efficiency, a large number of theoretical
plates are necessary.
Since N remains constant for different bands in a
chromatogram at the same conditions, it is predicted
that (W) increases proportionally with (tR) and N is a
useful measure of column to efficiency.
tR
W
Theoretical Consideration
The height equivalent to one theoretical plate (HETP)
HETP may be calculated from the value for N and
column length L
L

 HETP 

N

The value of HETP decreases as the efficiency of
column increases while the value for N is useful for
comparing the relative efficiencies of different
columns, the HETP is useful in assessing the varying
efficiency of the same column under different
condition.
Plate Height (h)
Theoretical Consideration
A more useful guide to column efficiency is plate
height (h), given by:
HETP
h
dp
where:
dp is the diameter of the stationary phase particles. This
value is useful for the comparison of different columns.
Asymmetry Factor (Af)
For practical reasons, the asymmetry factor is
measured at 10% of peak height.
where A is the distance
from peak front to peak
maximum and B is the
distance
from
peak
maximum to peak end.
B
Af 
A
Theoretical Consideration
Ideally, symmetry should be in the range 0.95-1.15.
With values below 1 is peak fronting.
With values above 1 there is peak tailing.
47
Theoretical Consideration
Degree Resolution (Rs)
The previous equation for resolution:
 t R 2  t R1 

Rs  2
 W 1  W2 
does not show how resolution is related to the
condition of separation and can not be used
directly to improve resolution. Therefore,
another equation for resolution is applied as a
function of column efficiency, N, selectivity, 
and capacity factor, K.
Rs 
N
4
   1  K 



   1  K 
Pharmaceutical applications of HPLC
Quantitative
analysis
of
a
chromatogram is the process of measuring
the peak response from the detector and
comparing the response to that of a known
calibration curve. A calibration curve is a
plot of a detector response (peak area
height) versus the concentration.
Calibration curve
Quantitative Analysis
Peak hight
External Standard Method
Concentration
0
10
100 g/mL
0
10
75 g/mL
0
10
50 g/mL
0
10
25 g/mL
0
10
10 g/mL
0
10
5 g/mL
0
10
Unknown
Calibration curve
Peak hight ratio
Quantitative Analysis
Internal Standard Method
Concentration ratio
Internal
Internal
Standard
Standard
Compound
Compound
0
10
100 g/mL
0
10
75 g/mL
0
10
50 g/mL
0
10
25 g/mL
0
10
10 g/mL
0
10
5 g/mL
0
10
Unknown
Properties of internal standards
Pharmaceutical applications of HPLC
Pharmaceutical applications of HPLC
Pharmaceutical applications of HPLC
Pharmaceutical applications of HPLC
Pharmaceutical applications of HPLC
Pharmaceutical applications of HPLC
Pharmaceutical applications of HPLC
59
Pharmaceutical applications of HPLC
Pharmaceutical applications of HPLC