Pharmaceutical AnalysisPharmaceutical Analysis
ChromatographyChromatography
Petra University Petra UniversityFaculty of Pharmacy & Medical ScienceFaculty of Pharmacy & Medical Science
Dr. Wael Abu Dayyih1
C Chrormatography
مقدمةهذه المحاضرة البسيطة تندرج تحت قسم )الكيمياء التحليلية( أو باألخص بقسم )التحليل باستخدام األجهزة( وهي مختلفة قليال عن الكيمياء التحليلية مع أنهما يتبعان نفس المبادئ العلمية, وهي من المواد المهمة جدا في األقسام الطبية
السيما منها الصيدالنية, تعمل على دراسة الجوانب المختلفة مما يسمى )الكروماتوجراف( وهي آلية تحليل المادة الى مكوناتها الدقيقة للتعرف على
محتوياتها, وهذه طريقة قديمة حديثة مهمة جدا ويعتمد عليها بشكل كبير جدا في مختبرات األدوية والتشاخيص وتحديد السموم, وفي هذا الجزء من سلسلة
المحاضرات نتناول دراسة سريعة حول تقنية حديثة نوعا ما من الكروماتوجراف, تستخدم فيها المضخات لضخ المادة المذيبة إلى داخل السائل الذي يحتوي على
المواد المراد قياس نسبها ونوعيتها, وتستخدم فيها األجهزة المعملية الحديثة لهذا الموضوع والجميل في هذه التقنية انها استحدثت األساليب القديمة
وأضافت عليها التكنولوجيا الحديثة والكمبيوترات السريعة وباستخدام المعادالت الرياضية الطبيعية في علوم الجبر , تمكن الباحثون من استخالص آلية جميلة
تساعد على استخالص المواد المكونة للمادة الواحدة, ومقارنتها ويبعضها البعض ومقارنتها أيضا برسوم جبرية ثابتة القياس بوجود نفس الظروف, مما يؤدي الى
سرعة في تحديد نوعية المواد وماهيتها بشكل دقيق
الناس موتى و أهل العلم أحياء و الناس مرضى و هم فيها أطباء و الناس أرض و أهل العلم فوقهم مثل السماء و ما في النور ظلماء و زمرة العلم رأس الخلق كلهم و سائر الناس في التمثال أعضاء
تعتبر طرق الكروماتوغرافيا أكثر الطرق استعماال في مختبرات التحليل وفي إحصائية في مجلة1980
%من األبحاث المنشورة استخدمت طرق التحليل 27أن طرق التحليل المعتمدة * % في مجاالت تحليل27% من ال 70الكرومتوغرافي وان
األدوية وتعتبر من أكثر الطرق اتساعا وانتشارا بين طرق التحليل والسبب هو أن هذه الطرق تسمح للمحلل الكيميائي بتمرير المادة المذابة
في الطور السائل عبر العمود المملئ بالطور الثابت حيث يحدث الفصل عن طريق حصول أالف العمليات الدقيقة بداخل العمود
*Official Methods in Analysis
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IntroductionIntroduction
• 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.
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IntroductionIntroduction• 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 phase could be thought of as the air.
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IntroductionIntroduction• 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.
• The two are not mutually exclusive.5
Chromatography TermsChromatography 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.
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Plotted on the x-axis is Plotted on the x-axis is the retention time and the retention time and plotted on the y-axis a plotted on the y-axis a signal (for example signal (for example obtained by a obtained by a spectrophotometer, spectrophotometer, mass spectrometer or mass spectrometer or a variety of other a variety of other detectors) detectors) corresponding to the corresponding to the response created by response created by the analytes exiting the the analytes exiting the system. system.
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Chromatography TermsChromatography 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)
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Chromatography TermsChromatography 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).
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Chromatography TermsChromatography 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.
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Chromatography TermsChromatography 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.
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Chromatography TermsChromatography 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.
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Chromatography theoryChromatography 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.
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RetentionRetention
• 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:
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RetentionRetention
• 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.
solventbymovedcedis
compoundbymovedcedisR f tan
tan
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RetentionRetention
• 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.
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Plate TheoryPlate 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:
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Plate TheoryPlate 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''.
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Techniques by chromatographic Techniques by chromatographic bed shapebed shape
Column ChromatographyColumn 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).
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Column ChromatographyColumn Chromatography
• A diagram of a standard column chromatography and a flash column chromatography setup20
What is ChromatographyWhat 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.
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What is ChromatographyWhat 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
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What is ChromatographyWhat is Chromatography
• Liq/Liq (Partition) Liquid• Liq/Sol (Adsorption) Chromatography
• Gas/Liq (Partition) Gas• Gas/Sol (Adsorption) Chromatography
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Chromatography modesChromatography 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.
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HPLCHPLC
• 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»
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HPLCHPLC
• 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
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HPLCHPLC• 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
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Table 1: Classification of column Table 1: Classification of column chromatographic methods chromatographic methods
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Chromatography BasicsChromatography Basics
Time or Volume
Typical ChromatogramD
etec
tor
Res
pons
e
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Chromatography BasicsChromatography Basics
Time or Volume
tm = time for mobile phase to travel length of column (dead time)
Det
ecto
r R
espo
nse
tr = retention time
t’ = adjusted retention time
= tr - tm
=t’r2 t’r1=relative retention (selectivity factor)
tr = injection time
tmt’r1
tr1
tr2
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Chromatography BasicsChromatography 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 UN-RETARDED MOLECULE TO FLOW THROUGH THE COLUMN (VOID TIME)(DEAD TIME) BUT THE LENGTH OF TIME IT TAKES ARETARDED COMPOUND TO PASS THROUGH COLUMN tr
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Partition RatiosPartition 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.
m
sc
cK
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Partition RatiosPartition 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
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Capacity Factor, kCapacity 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
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Capacity Factor, kCapacity Factor, k’’
• A measure of retention: An experimental parameter widely used to describe solute rates on columns.
• (higher k’= greater solute retention)
• If k’ ≤1: elution too rapid for accurate determination of tr.
• If k’> approx. 10: elution too slow to be participle.
• Preferred range for k’ is approximately 1-5.
This is an experimentally easy way to determine k’.
m
s
m
s
moles
moles
V
Vkk '
m
mr
m
s
t
tt
t
tk
'
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fraction of timefraction of time
• What fraction of time does the solute spends in mobile phase?
• q = fraction of solute in mobile phase
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Chromatography BasicsChromatography Basics
'
1
1
1
1
kfactorcapacityV
VK
VV
Kq
VCVC
q
VCVC
VC
molesmoles
molesq
m
s
m
s
mm
ss
ssmm
mm
ms
m
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Chromatography BasicsChromatography Basics
• Fraction of time solute spends in mobile phase:
• Larger k’ means greater retention times• Fraction of time solute spends in stationary phase
= (1-q)
'1
')1(
k
kq
'1
1
kq
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Chromatography BasicsChromatography Basics
• Rate of travel of solute molecule through column (v’):
Linear Flow Rate (cm/min)
V’ = v (fraction of time in mp)
'1
1
1
1'
kv
V
VK
vv
m
s
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Chromatography BasicsChromatography Basics
• Retention time tr: time it takes solute to go from beginning to end of column.
travelsoluteofrate
Ltr
Column length
m
s
r
VV
Kv
Lt
1
1
v
Ltm
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Chromatography BasicsChromatography Basics
• Retention time tr:
• Retention volume (Vr): multiply retention tme (tr) by volumetric flow rate, F (Vm/tm)
)'1()1( ktV
VKtt m
m
smr
)()1( smm
smr KVV
V
VKVV
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Chromatography BasicsChromatography Basics
Time or Volume
Typical ChromatogramD
etec
tor
Res
pons
e
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Chromatography BasicsChromatography 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
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Efficiency of SeparationEfficiency of Separation
• Two factors affect how well two components are separated:– Difference in retention time– Peak widths
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Efficiency of SeparationEfficiency of Separation
• Solutes in a column spread into a Gaussian profile:
• Gaussian peak shape:
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Efficiency of SeparationEfficiency of Separation
• The resolution (separation) of two solutes:
tr = difference between retention times of two peaks = (tr2-tr1)
• Wavg= average of the peak widths at baseline (4)
avg
r
avg
r
W
V
W
tresolution
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• 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.
• Usually competition between speed and resolution.49
Efficiency of SeparationEfficiency 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
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Efficiency of SeparationEfficiency of Separation
• N is specific for each solute on a given column• Increasing retention time increases N
2/1
2
2
2
2
2 55.516
w
t
w
ttN rrr
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Efficiency of SeparationEfficiency of Separation
• N’s relation to Resolution (R):
'
'2
1
1
4 avgk
kNR
= relative retention (selectivity factor)
1
2
1
2
1
2
'
'
'
'
K
K
k
k
t
t
r
r 52
Efficiency of SeparationEfficiency of Separation
• N depends upon the length of the column • Independent of the column length is the Height
Equivalent of a Theoretical Plate
• As HETP , resolution increases (N)
2
2
16 rt
Lw
N
LHETP
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Why Bands SpreadWhy Bands Spread
• Band broadening
• Causes of band broadening:– Eddy diffusion: (A)– Longitudinal diffusion: (B)– Resistance to mass transfer (RMT): (C)
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Why Bands SpreadWhy Bands Spread
• Eddy diffusion (not simple diffusion):
• HA = A:A = constant, depends on size of particles
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Why Bands SpreadWhy 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 SpreadWhy Bands Spread
• Resistance to mass transfer (RMT):
HC = Cv: C = constant, v= flow rate
Decrease HC by decreasing v.58
Why Bands SpreadWhy Bands Spread
• Van Deemter Equation:
HETP = HA + HB + HB
HETP = A + (B/v) + Cv
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