Pharmaceutical Analysis
Chromatography
Petra University
Faculty of Pharmacy & Medical Science
1
Dr. Wael Abu Dayyih
‫مقدمة‬
‫هذه املحاضرة البسيطة تندرج تحت قسم (الكيمياء التحليلية) أو باألخص بقسم (التحليل باستخدام األجهزة) وهي مختلفة قليال عن‬
‫الكيمياء التحليلية مع أنهما يتبعان نفس املبادئ العلمية‪ ,‬وهي من املواد املهمة جدا في األقسام الطبية السيما منها الصيدالنية‪ ,‬تعمل‬
‫على دراسة الجوانب املختلفة مما يسمى (الكروماتوجراف) وهي آلية تحليل املادة الى مكوناتها الدقيقة للتعرف على محتوياتها‪ ,‬وهذه‬
‫طريقة قديمة حديثة مهمة جدا ويعتمد عليها بشكل كبيرجدا في مختبرات األدوية والتشاخيص وتحديد السموم‪ ,‬وفي هذا الجزء من‬
‫سلسلة املحاضرات نتناول دراسة سريعة حول تقنية حديثة نوعا ما من الكروماتوجراف‪ ,‬تستخدم فيها املضخات لضخ املادة املذيبة إلى‬
‫داخل السائل الذي يحتوي على املواد املراد قياس نسبها ونوعيتها‪ ,‬وتستخدم فيها األجهزة املعملية الحديثة لهذا املوضوع والجميل في هذه‬
‫التقنية انها استحدثت األساليب القديمة وأضافت عليها التكنولوجيا الحديثة والكمبيوترات السريعة وباستخدام املعادالت الرياضية‬
‫الطبيعية في علوم الجبر ‪ ,‬تمكن الباحثون من استخالص آلية جميلة تساعد على استخالص املواد املكونة للمادة الواحدة‪ ,‬ومقارنتها‬
‫ويبعضها البعض ومقارنتها أيضا برسوم جبرية ثابتة القياس بوجود نفس الظروف‪ ,‬مما يؤدي الى سرعة في تحديد نوعية املواد وماهيتها‬
‫بشكل دقيق‬
‫و الناس مرض ى و هم فيها أطباء‬
‫الناس موتى و أهل العلم أحياء‬
‫و الناس أرض و أهل العلم فوقهم مثل السماء و ما في النور ظلماء‬
‫و سائرالناس في التمثال أعضاء‬
‫و زمرة العلم رأس الخلق كلهم‬
‫تعتبرطرق الكروماتوغرافيا أكثرالطرق استعماال في مختبرات التحليل وفي إحصائية ‪ 1980‬في مجلة‬
‫طرق التحليل املعتمدة * أن ‪%27‬من األبحاث املنشورة استخدمت طرق التحليل الكرومتوغرافي وان ‪ %70‬من ال ‪ %27‬في مجاالت تحليل‬
‫األدوية وتعتبر من أكثرالطرق اتساعا وانتشارا بين طرق التحليل والسبب هو أن هذه الطرق تسمح للمحلل الكيميائي بتمرير املادة املذابة‬
‫في الطور السائل عبرالعمود اململئ بالطور الثابت حيث يحدث الفصل عن طريق حصول أالف العمليات الدقيقة بداخل العمود‬
‫‪*Official Methods in Analysis‬‬
‫‪Chrormatography‬‬
‫‪C‬‬
‫‪2‬‬
Introduction
• Chromatography (from Greek χρῶμα chroma
"color" and γράφειν graphein "to write") is the
collective term for a set of laboratory techniques
for the separation of mixtures. It involves
passing a mixture dissolved in a "mobile phase"
through a stationary phase, which separates the
analyte to be measured from other molecules in
the mixture and allows it to be isolated.
3
Introduction
• For those new to separation science, an analogy
which is sometimes useful is to suppose a
mixture of bees and wasps passing over a flower
bed.
• The bees would be more attracted to the flowers
than the wasps, and would become separated
from them.
• If one were to observe at a point past the flower
bed, the wasps would pass first, followed by the
bees.
• In this analogy, the bees and wasps represent
the analytes to be separated, the flowers
represent the stationary phase, and the mobile
4
phase could be thought of as the air.
Introduction
• The key to the separation is the differing affinities among
analyte, stationary phase, and mobile phase.
• The observer could represent the detector used in some
forms of analytical chromatography.
• A key point is that the detector need not be capable of
discriminating between the analytes, since they have
become separated before passing the detector.
• Chromatography may be preparative or analytical.
• Preparative chromatography seeks to separate the
components of a mixture for further use (and is thus a
form of purification).
• Analytical chromatography normally operates with
smaller amounts of material and seeks to measure the
relative proportions of analytes in a mixture.
•5 The two are not mutually exclusive.
Chromatography Terms
• The analyte is the substance to be separated
during chromatography.
• Analytical chromatography is used to
determine the existence and possibly also the
concentration of analyte(s) in a sample.
• A bonded phase is a stationary phase that is
covalently bonded to the support particles or to
the inside wall of the column tubing.
• A chromatogram is the visual output of the
chromatograph. In the case of an optimal
separation, different peaks or patterns on the
chromatogram
correspond
to
different
components
of
the
separated
mixture.
6
Plotted on the x-axis is
the retention time and
plotted on the y-axis a
signal (for example
obtained by a
spectrophotometer,
mass spectrometer or
a variety of other
detectors)
corresponding to the
response created by
the analytes exiting the
system.
7
Chromatography Terms
 A Chromatograph takes a chemical misture
carried by liquid or gas and separates it into
component parts as a result of differential
distributions of the solutes as they flow around
or over the stationary phase.(instrument)
 Chromatography is a physical method of
separation in which the components to be
separated are distributed between two phases,
one of which is stationary (stationary phase)
while the other (the mobile phase) moves in a
definite direction.(method)
8
Chromatography Terms
• The effluent is the mobile phase leaving the
column.
• An immobilized phase is a stationary phase
which is immobilized on the support particles, or
on the inner wall of the column tubing.
• The mobile phase is the analyte and solvent
mixture which travels through or along the
stationary phase, in a definite direction. It may
be a liquid (LC), a gas (GC), or a supercritical
fluid (supercritical-fluid chromatography).
9
Chromatography Terms
 Preparative chromatography is used to
nondestructively purify sufficient quantities
of a substance for further use, rather than
analysis.
 The retention time is the characteristic
time it takes for a particular molecule to
pass through the system under set
conditions.
10
Chromatography Terms
 The sample is the mixture consisting of a
number of components the separation of
which
is
attempted
on
the
chromatographic system as they are
carried or eluted by the mobile phase.
 The solute refers to the sample
components in partition chromatography.
 The solvent refers to the liquid stationary
phase in partition chromatography.
11
Chromatography Terms
 The stationary phase is the substance
which is fixed in place for the
chromatography procedure and is the
phase to which solvents and the analyte
travels through or binds to. Examples
include the silica layer in thin layer
chromatography.
12
Chromatography theory
• Chromatography is a separation method that
exploits the differences in partitioning behavior
between a mobile phase and a stationary
phase to separate the components in a mixture.
• Components of a mixture may be interacting
with the stationary phase based on charge,
relative solubility or adsorption.
• There are two theories of chromatography, the
plate and rate theories.
13
Retention
• The retention is a measure of the speed at which
a substance moves in a chromatographic
system. In continuous development systems like
HPLC or GC, where the compounds are eluted
with the eluent, the retention is usually
measured as the retention time Rt or tR, the time
between injection and detection.
• In interrupted development systems like TLC the
retention is measured as the retention factor Rf,
the run length of the compound divided by the
run length of the eluent front:
14
Retention
dis tan ce moved by compound
Rf 
dis tan ce moved by solvent
• The retention of a compound often differs
considerably
between
experiments
and
laboratories due to variations of the eluent, the
stationary phase, temperature, and the setup. It
is therefore important to compare the retention
of the test compound to that of one or more
standard compounds under absolutely identical
conditions.
15
Retention
• During the chromatographic process the analyte
experiences zone broadening as a result of
diffusion. Two analytes with different retention
times yet with large broadening do not resolve
and this is why in any chromatographic system
broadening needs to be minimized. This is done
by selecting the proper stationary and mobile
phase, the eluent velocity, the track length and
temperature. The Van Deemter's equation gives
an ideal eluent velocity taking into account
several physical parameters.
16
Plate Theory
• The plate theory of chromatography was
developed by Archer John Porter Martin and
Richard Laurence Millington Synge. The plate
theory describes the chromatography system,
the mobile and stationary phases, as being in
equilibrium. The partition coefficient ''K'' is based
on this equilibrium, and is defined by the
following equation:
17
Plate Theory
• ''K'' is assumed to be independent of
concentration, and can change if experimental
conditions
are
changed,
for
example
temperature is increased or decreased. As ''K''
increases, it takes longer for solutes to separate.
For a column of fixed length and flow, the
retention time (tR) and retention volume (Vr) can
be measured and used to calculate ''K''.
18
Techniques by chromatographic
bed shape
Column Chromatography
• Column chromatography is a separation
technique in which the stationary bed is within a
tube. The particles of the solid stationary phase
or the support coated with a liquid stationary
phase may fill the whole inside volume of the
tube (packed column) or be concentrated on or
along the inside tube wall leaving an open,
unrestricted path for the mobile phase in the
middle part of the tube (open tubular column).
19
Column Chromatography
• A diagram of a standard column chromatography and a
20 flash column chromatography setup
What is Chromatography
• Method to separate components in a
mixture based on different Distribution
coefficients between the two immiscible
phases.
• Same principle as solvent (extraction), but
one phase is fixed (stationary) and the
other phase is moving (mobile).
• Stationary phases are most commonly
coated or packed in a column.
21
What is Chromatography
• Chromatography is categorized on the basis of
interaction between solute and stationary phase.
Stationary phase either liquid or solid.
• It is also classified into:
– Planar chromatography – flat stationary phase,
mobile phase moves through capillary action or
gravity.
– Column chromatography – tube of stationary phase,
mobile phase moves by pressure or gravity
• Mobile phase either gas or liquid so we have
– Liquid Chromatography and
– Gas Chromatography
22
What is Chromatography
• Liq/Liq
• Liq/Sol
(Partition)
(Adsorption)
Liquid
Chromatography
• Gas/Liq
• Gas/Sol
(Partition)
(Adsorption)
Gas
Chromatography
23
Chromatography modes
• Other modes of chromatography (Table 1)
– Ion-exchange:
separates
charges
species
– Size exclusion or Gel Permeation:
separated according to molecular size.
– Affinity: separates on the basis of
antibody-antigen,
enzyme-substrate
interactions.
24
HPLC
• Normal phase HPLC: the packing
material in the column is usually
simple unmodified silica gel « the
natural silica is polar polymer with the
main functional group known as the
silanol group in this case we have a
polar stationary phase»
25
HPLC
• Reversed phase HPLC : the stationary
phase is a modified silica and its also
called BONDED PHASE ►its made by
reacting the silanol group of natural silica
with hydrophobic groups like
OCTADECANE (C18)to form polymer with
lipophylic character=
ODS→OCTADECYLSILANE
26
HPLC
• Mobile phase RP-HPLC
• The mobile phase is usually mixture of
water plus other organic solvents like:
• - methanol
• - acetonitrile
• - tetrahydroforan THF
NOTE … INCREASING THE PERCENTAGE OF
WATER/ORGANIC COMPONENTS LEAD TO
INCREASE IN THE RETENTION TIME OF
ANALYTE
27
Table 1: Classification of column
chromatographic methods
28
29
Chromatography Basics
Detector
Response
Typical Chromatogram
Time or Volume
30
Detector
Response
Chromatography Basics
tr = injection time
tr2
tr1
tm
t’r1
Time or Volume
tm = time for mobile phase to travel tr = retention time
length of column (dead time)
t’ = adjusted retention time
= 31tr - tm
=t’r2 t’r1=relative retention
(selectivity factor)
Chromatography Basics
• Mobile phase flow rate:
– Volumetric flow rate (F): ml/min
– Liner flow rate (v): cm/min (mm/min)
• Two ways to describe solute “retention”
– Retention time, tr
– Retention volume, Vr
• Vr = Ftr
• VOID VOLUME:(v0)=t0: IS THE LENGTH OF TIME TAKES AN UNRETARDED MOLECULE TO FLOW THROUGH THE COLUMN
(VOID TIME)(DEAD TIME) BUT THE LENGTH OF TIME IT TAKES
ARETARDED COMPOUND TO PASS THROUGH COLUMN tr
32
Partition Ratios
• Consider solute species A as
equilibrium
Amobile

Astationary
The equilibrium constant is:
Where:
K= partition ratio or partition
coefficent;
Cs, cm = concentrations of A in
stationary, mobile phases.
33
K
cs
cm
Partition Ratios
• Partition coefficient K = Cs/Cm
– C = Concentration of analyte
– s = stationary phase
– m = mobile phase
• Vs = volume of stationary phase
• Vm = volume of mobile phase
34
Capacity Factor, k’
• k’: WHICH IS A MEASURE OF THE
DEGREE TO WHICH IT PARTITIONS
(ADSORPTION) INTO THE STATIONARY
PHASE FROM THE MOBILE PHASE :
• k’=(tr-t0)/t0
35
Capacity Factor, k’
• A measure of retention: An experimental parameter
widely used to describe solute rates on columns.
• (higher k’= greater solute retention)
Vs moless
k' k

Vm molesm
ts tr  tm
k' 
tm
tm
This is an experimentally easy way
to determine k’.
• If k’ ≤1: elution too rapid for accurate determination of tr.
• If k’> approx. 10: elution too slow to be participle.
36
• Preferred range for k’ is approximately 1-5.
fraction of time
• What fraction of time does the solute
spends in mobile phase?
• q = fraction of solute in mobile phase
37
Chromatography Basics
molesm
CmVm
q

moless  molesm CmVm  CsVs
1
q
CsVs
1
CmVm
q
1
Vs
1 K
Vm
Vs
K
 capacity factor  k '
Vm
38
Chromatography Basics
• Fraction of time solute spends in mobile phase:
1
q
1 k'
• Larger k’ means greater retention times
• Fraction of time solute spends in stationary phase
= (1-q)
39
k'
(1  q ) 
1 k'
Chromatography Basics
• Rate of travel of solute molecule through column
(v’):
Linear Flow Rate (cm/min)
V’ = v (fraction of time in mp)
40


 1

 1 
  v
v'  v 

V
1  k ' 
1  K s 

Vm 
Chromatography Basics
• Retention time tr: time it takes solute to go from
beginning to end of column.
L
tr 
rate of solute travel
tr 
41
Column length
L


1
v
1  K Vs

Vm





L
tm 
v
Chromatography Basics
• Retention time tr:
Vs
t r  t m (1  K )  t m (1  k ' )
Vm
• Retention volume (Vr): multiply retention tme (tr)
by volumetric flow rate, F (Vm/tm)
Vs
Vr  Vm (1  K )  Vm ( KVs )
Vm
42
Chromatography Basics
Detector
Response
Typical Chromatogram
Time or Volume
43
Chromatography Basics
• Calculation of column efficiency:
• The efficiency of the column is assessed
from the width of the peak at half height
W1/2 and its retention time using the
following equation :
• N=5.54(tr/W1/2)2
•
NOTE… THE COMPOUND WITH THE LARGEST CAPACITY FACTOR
EMERGES LAST
44
Efficiency of Separation
• Two factors affect how well two
components are separated:
– Difference in retention time
– Peak widths
45
Efficiency of Separation
• Solutes in a column spread into a Gaussian
profile:
• Gaussian peak shape:
46
Efficiency of Separation
• The resolution (separation) of two solutes:
tr
Vr
resolution 

Wavg Wavg
• tr = difference between retention times of two
peaks = (tr2-tr1)
• Wavg= average of the peak widths at baseline
(4)
47
Efficiency of Separation
• Resolution : higher R. better separation
48
• This shows two ways of
improving resolution:
• (b) increased separation or
• (c) decreased band width.
• Details later on influencing
band width
• Note: If there was always an equilibrium distribution
between mobile and stationary phases, there would be
much less band broadening – but this would take
excessively long times.
49
• Usually competition between speed and resolution.
Efficiency of Separation
• Plate Theory:
– Treats separation in discrete stages, more
stages = more plates.
• Theoretical plates (N): a number indicating
how good a column is for a separation
• Plate theory: a theory that was made to
explain the mechanism of separation of
analyses on chromatographic system
(imaginary unit) these plates are known as
theoritical plates N=16(tr/w)2
50
Efficiency of Separation
t
2
r
2
2
r
2
16t
5.55t
N



w
w1/ 2
2
r
• N is specific for each solute on a given column
• Increasing retention time increases N
51
Efficiency of Separation
• N’s relation to Resolution (R):
'

N    1  k 2
R


'
4    1  k avg




•  = relative retention (selectivity factor)
52
t 'r 2 k '2 K 2



t 'r1 k '1 K1
Efficiency of Separation
• N required to obtain a certain resolution:
53
Efficiency of Separation
• N depends upon the length of the column
• Independent of the column length is the Height
Equivalent of a Theoretical Plate
2
L Lw
HETP  
2
N 16t r
• As HETP , resolution increases (N)
54
Why Bands Spread
• Band broadening
• Causes of band broadening:
– Eddy diffusion: (A)
– Longitudinal diffusion: (B)
– Resistance to mass transfer (RMT): (C)
55
Why Bands Spread
• Eddy diffusion (not simple diffusion):
• HA = A:A = constant, depends on size of
particles
56
Why Bands Spread
• Longitudinal diffusion: solute [ ] is lower at
the edges of a band; solute diffuses to the
edges.
HB = B/v: B= constant, v= flow rate
Decrease HB by increasing v.
57
Why Bands Spread
• Resistance to mass transfer (RMT):
HC = Cv: C = constant, v= flow rate
Decrease HC by decreasing v.
58
Why Bands Spread
• Van Deemter Equation:
HETP = HA + HB + HB
HETP = A + (B/v) + Cv
59
Why Bands Spread
• Van Deemter Plot:
60