عرض تحليل الي م3_095500.pptx

Chromatographic separations
High Performance Liquid Chromatography
HPLC
Instrumental Analysis
Pressure
precision

To understand :
3. and the type of information it provides
1. the spectrometric and chromatography
techniques,
2. the theoretical concepts behind each
method,

Introduction Course syllabus and course policy.
Introduction to
chromatography
1.1.1 Introduction to Chromatography
1.1.2 What Is HPLC? /
1.1.3 A Brief History
1.1.4 Advantages and Limitations
1.2 -Modes of HPLC /
1.2.1 Normal-Phase Chromatography (NPC) /
1.2.2 Reversed-Phase Chromatography (RPC)
1.2.3 Ion-Exchange Chromatography (IEC) /
1.2.4 Size-Exclusion Chromatography (SEC)
1.2.5 Other Separation Modes /

2.2 Basic Terms and Concepts /
2.2.1 Retention Time (tR), Void Time (tM), Peak Height (h),and Peak Width (wb) /
2.2.2 Retention Volume (VR), Void Volume (VM), and Peak Volume
2.2.3 Retention Factor (k)
2.2.4 Separation Factor (α) /
2.2.5 Column Efficiency and Plate Number (N) /
2.2.6 Peak Volume /
2.2.7 Height Equivalent to a Theoretical Plate or Plate Height (HETP or H) /
2.2.8 Resolution (Rs) /
2.2.9 Peak Symmetry: Asymetry Factor (As) and Tailing Factor (Tf) /
2.3 Mobile Phase /
2.3.1 General Requirements /
2.3.2 Solvent Strength and Selectivity /
2.3.3 Buffers /
2.3.4 Acidic Mobile Phases /
2.3.6 High pH Mobile Phase /
2.3.7 Other Operating Parameters: Flow Rate (F) and Column Temperature (T) /
2.4 The Resolution Equation /

Electromagnetic
radiation
The interaction of electromagnetic
radiation with molecules, Double bond
equivalent
UV-Vis
spectrophotometry
2.1 Instrumentation
2.2 Selection Rules and the Beer-
Lambert Law
2.3 Chromophores
2.4 Applications of UV Spectroscopy
IR
spectrophotometry
3.1 Instrumentation
3.2 Selection Rules and Hooke's Law
3.3 Characteristic Group Vibrations
IRproblemsolving

Chromatography
is a method by which a mixture is separated
by distributing its components between two
phases
stationary phase and mobile phase

Chromatography Analytical technique
Chromatograph Instrument
Chromatogram
Chromatographer Person

Based on physical nature of the mobile phase:
a) Liquid Chromatography (LC),
b) Gas Chromatography (GC),
c) Supercritical Fluid Chromatography (SFC)
a) Liquid Chromatography (LC),
2. Thin-layer chromatography (TLC)
1. Paper chromatography ( PC)
3. HPLC
Classification of Chromatographic Separations

1. Paper chromatography ( PC) 2. Thin-layer chromatography (TLC)
3. HPLC (Elution)

b) Gas Chromatography (GC),

‫الحرجة‬ ‫درجته‬ ‫تبلغ‬ ‫الذي‬ ‫الماء‬
374
‫مئوية‬ ‫درجة‬
.
‫إلى‬ ‫النقطة‬ ‫تلك‬ ‫عند‬ ‫الضغط‬ ‫ويصل‬
218
‫جوي‬ ‫ضغط‬
.
‫الماء‬ ‫في‬ ‫الحرجة‬ ‫النقطة‬
‫الكربون‬ ‫أكسيد‬ ‫ثاني‬
‫الحرجة‬ ‫درجته‬ ‫تبلغ‬ ‫الذي‬
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‫مئوية‬ ‫درجة‬
‫الحرج‬ ‫وضغطة‬
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Applications of TLC
• Used to determine impurities in pharmaceutical raw materials and formulated products.
• Often used as a basic identity check on pharmaceutical raw materials.
• Potentially useful in cleaning validation, which is part of the manufacture of
pharmaceuticals.
GC :Applications
Limit tests for solvent residues and other volatile impurities in drug substances.
• Sometimes used for quantification of drugs in formulations, particularly if the drug
lacks a chromophore.
• Characterisation of some raw materials used in synthesis of drug molecules.
• Characterisation of volatile oils (which may be used as excipients in formulations),
proprietary cough mixtures and tonics, and fatty acids in fixed oils.
• Measurement of drugs and their metabolites in biological fluids.

Principle of High Performance Liquid Chromatography
1. The sample (the solutes) is dissolved in
a mobile phase ( is called the “eluent”),
which may be a gas, a liquid, or a
supercritical fluid.
2. The mobile phase is then forced
through an immiscible stationary
phase ( sorbents packed inside a
column).
3. Differences in the interactions between the
components of the sample and stationary and
mobile phases enable separation.

(b) Microscopic representation of the
partitioning process of analyte molecules
A and B into the stationary phase bonded
to a spherical solid support.
(a) Schematic of the chromatographic
process showing the migration of two
bands of components down a column.
(c) A chromatogram plotting the
signal from a UV detector displays
the elution of components A and B
Principle of High Performance Liquid Chromatography

Mikhail Tswett, who separated the chlorophyll into layers of different colors
on chalk (CaCO3) packed in glass columns in 1903.
A Brief History

Fields of used of HPLC
Biochemical
Carbohydrate, lipids, nucleic and amino acids, proteins.
Pharmaceuticals
antibiotics, sedatives , steroids , analgesics
Food products
Artificial sweeteners, antioxidants, aflatoxins , additives .
Industrial chemistry
Condensed aromatics , surfactants , propellants , dyes
Pollutants
Pesticides, herbicides, phenols, polychlorinated biphenyls.
Forensic science
Drugs , poisons , blood alcohol , narcotics
Clinical chemistry
Bile acids , drug metabolites , urine extracts , estrogens

Amenable to diverse samples
Advantages and Limitations of HPLC
Advantages
Rapid and precise quantitative analysis
Automated operation
High-sensitivity detection
Quantitative sample recovery

1 Normal-Phase Chromatography (NPC)
Mobile phase
Stationary phase
Phase
non-polar (hydrophobic)
lipophilic
Polar
(hydrophilic)
Polarity
Organic solvent
Silica +
Silanol groups
Material
non- Polar analyte
polar analyte
Interaction
with
non- Polar or the least polar compounds
First eluted
nonpolar compounds and isomers
Analysis
Silanol groups (Si-OH)
silica support
MODES OF HPLC or Types of column HPLC

Figure 4. A normal-phase HPLC chromatogram of a palm olein sample showing
the separation of various isomers of vitamin E.

In reversed phase chromatography, the most polar compounds elute first with the
most nonpolar compounds eluting last. The mobile phase is generally a binary mixture
of water and a miscible polar organic solvent like methanol, acetonitrile or THF.
Retention increases as the amount of the polar solvent (water) in the mobile phase
increases. Reversed-phase chromatography, a partition mechanism, is typically used
for separations by non-polar differences.
n normal-phase chromatography, the least polar compounds elute first and the
most polar compounds elute last. The mobile phase consists of a nonpolar solvent
such as hexane or heptane mixed with a slightly more polar solvent such
as isopropanol, ethyl acetate or chloroform. Retention decreases as the amount of
polar solvent in the mobile phase increases.
Normal phase chromatography, an adsorptive mechanism, is used for the analysis
of solutes readily soluble in organic solvents, based on their polar differences such
as amines, acids, metal complexes, etc..

octadecyl (C18) groups
silica support
2 Reversed-Phase Chromatography (RPC)
Mobile phase
Stationary phase
Phase
polar (hydrophilic).
Non-polar
(hydrophobic)
Polarity
mixture of methanol or
acetonitrile with water
Silica +
Octdecyl groups
Material
Polar analyte
Non- Polar analyte
Interaction
with
Polar first and nonpolar last
First eluted
analysis of polar (water-soluble), medium-
polarity, and some nonpolar analytes.
Analysis

Figure 5. A reversed-phase HPLC chromatogram of three organic components

3 Ion-Exchange Chromatography (IEC)
Cationic exchange (sulfonate)
ionic
Analytes
( Cationic )
analysis of ions and biological components such as amino acids, proteins/peptides,
and polynucleotides.

based on the analyte’s molecular size.
4 Size-Exclusion Chromatography (SEC)
a small molecule can penetrate
the pores and migrates more
slowly down the column.
A large molecule is excluded from the
pores and migrates quickly,

Affinity chromatography : Based on a receptor/ligand interaction in which immobilized
ligands (enzymes, antigens, or hormones) on solid supports are used to isolate selected
components from a mixture.
Chiral chromatography: For the separation of enantiomers using a chiral-
specific stationary phase. Both NPC and RPC chiral columns are available
Hydrophilic interaction chromatography: using a polar stationary phase such as
silica or ion-exchange materials with polar mobile phases of organic solvents and
aqueous buffers. It is most commonly used to separate polar analytes and hydrophilic
peptides.

Silanol formed by heating the silica particles-in dilute acid for a day or two so as to
generate the reactive silonal group :

Predict the order of elution, from first to last, of the following steroids
from an ODS column with methanol/water (70:30) as the mobile phase.
Answer: estradiol, nandrolone, testosterone, methyltestosterone.
Lipophilic methyl
group
lipophilicity
polarity

k = 0 is a component that is unretained by the stationary phase
k > 20 indicates that the component is highly retained.
When k is < 1.0, separation is poor
When k is > 20, separation is slow
(component is highly retained)
When k is = 1-10, separation is optimum

Since all of 𝑘’ values for 2 and 3 lie in the
preferred range of 2-10, the peaks are suitable
for quantitation , but, peak 1 is not.

In a chromatographic analysis of a drug sample , the drug
elutes with a retention time of 7.63 min. The column’s dead
time is 0.31 min. Calculate the capacity factor for the drug.

The selectivity factor ( α ) of a column is defined as the degree of separation
between successive peaks
For the two A and B components, α is defined as:
𝑘′𝐵 and 𝑘′𝐴 are the
retention factors
or Separation Factor
A chromatogram of two peaks with a selectivity factor (α) of 1.3.
Selectivity must be >1.0 for peak separation.

high selectivity
high selectivity
Low selectivity
low selectivity

Selectivity is dependent on many factors that affect K such as :
 the nature of the stationary phase
 the mobile phase composition
 properties of the solutes.
All of these factors may be changed during method development to increase the
separation of key analytes in the sample.

Calculate the selectivity factor (α) for the peak pairs of 1,2 and 3,4 and 5,6 in the
chromatogram shown below.
tR (in min) from left to right are: 0.20, 0.25, 0.53, 0.83, 1.52 and 2.25. and for the
unretained peak is 0.08 min

Analgesic acetaminophen and narcotic analgesics were separated using ultra-high
performance liquid chromatography (UHPLC) on an ultra C18 column and the following
chromatogram was obtained.
Calculate the selectivity factor (α) for the peak pairs of 2,3 and 3,4 and 4,5.

In UHPLC, particle sizes less than 2µm can be used,
providing better separation than HPLC where particle size
is limited to 5µm. These smaller particles require higher
pump pressures (100MPa vs.40 MPa, making this
technique very efficient with fast analysis and higher
resolution

Column Efficiency and Plate Number (N)

An efficient column produces sharp peaks and can
separate many sample components in a relatively short
time.
Column Efficiency and Plate Number (N)
broad peak
Large width
sharp beak
Small width

The band broadening inside the column is fundamental to all chromatographic
processes.
The plates number (N) is a measure of the efficiency of the column.
N = 20,000 plates OR N = 15,000 plates
N ↑ , Effi. ↑ , Wb ↓
The bigger the N , the better column efficiency
W½ at half height

high selectivity
high selectivity
Low selectivity
poor selectivity
High Column efficiency High Column efficiency
low Column efficiency
poor Column efficiency
Peaks far from each other and narrow Peaks are partial overlapping each
other and narrow
Peaks are far from each
other, and wide
Peaks are fully overlapping
each other and wide,

Resolution (Rs)
tR1
tR2
The goal of most HPLC analyses is the separation of one or more analytes in the
sample from all other components present.
Resolution (Rs) is a measure of the degree of separation of two adjacent analytes.

Figure 2.7 is a graphic representation of resolution for two peaks with
Rs ranging from 0.6 to 2.0.
Note that :
Rs = 0 indicates no separation.
Rs = 0.6 indicates a slight partial separation.
Rs = 1 indicates that a partial separation and is the minimum separation required

Rs = 1.5 indicates baseline separation.
Ideally, the goal of most HPLC methods is to achieve baseline separation
(RS = 1.5 – 2.0) for all key analytes

Peak Symmetry Asymmetry Factor (As) and Tailing Factor (Tf)
The asymmetry factor (As) is used to measure the degree of peak symmetry
As defined at peak width of
10% of peak height (W0.1).
Tf defined at the peak width
of 5% of peak height (W0.05)

Tf = 1 The peak is completely symmetric
Tf  1 Peak tailing
Tf < 1 Peak fronting
For most peaks (0.5 < Tf < 2)

t-Butylbenzene is a neutral and hydrophobic molecule, which elutes much later
but with excellent peak symmetry.
Pyridine is a base and exhibits peak tailing due to hydrophilic interaction with
residual silanol groups in addition to the partitioning process with the C-18 bonded
phase

MOBILE PHASE
The mobile phase is the solvent that moves the solute (analyte)
through the column
In HPLC, the mobile phase interacts with both the solute
and the stationary phase and has a powerful influence on
solute retention and separation.

• low viscosity, low toxicity, and non-flammability
General Requirements of mobile phase(solvents )
• High solubility for the sample components
• Noncorrosive to HPLC system components
• High purity, low cost, UV transparency

Increasing of
polarity
n-Hexane
iso-Octane （iso-Oct）
Chloroform （CHCl3）
Dichloromethane （CH2Cl2）
Ethylacetate （AcOEt）
Isopropylalchol （IPA）
Tetrahydrofuran （THF）
Dioxane
Acetonitrile（CH3CN）
Ethanol （EtOH）
Methanol （MeOH）
Amines
Acids
water
Some of solvent strengths under normal phase conditions

Solvent Strength and Selectivity
Solvent strength the ability of a solvent to elute solutes from a column.
In NPC
water is a strong solvent
In RPC
water is a weak solvent
stationary phase is polar (hydrophilic )
Nonpolar hexane is a weak solvent
stationary phase is nonpolar ( hydrophobic)
because it is a poor solvent for non strongly
H-bonding organics.
Nonpolar hexane is a strong solvent

Figure 2.10 shows a series of six chromatograms to illustrate the effect of solvent strength in RPLC
nitrobenzene (NB) and propylparaben (PP)
At 100% ACN, both components
are not retained by the column
and elute with a k close to zero.
At 60% ACN, the peaks are
slightly retained (k close to 1)
and are partially separated.

At 40% ACN ,The two components
merge back together.
At 30% ACN, the two
Components are well separated
At 20% ACN, propylparaben is
highly retained with a k of 31.
nitrobenzene and propylparaben

increase N (efficiency) by:
•Increasing column length
•Decreasing particle size
•Increasing temperature
Change α )selectivity( by:
•Changing column stationary phase
•Changing mobile phase pH
•Changing mobile phase solvent(s)
increase k (retention) by:
•Using a weaker solvent (changing polarity)
•Changing the ionization (polarity) of the analyte by changing pH
•Using a stronger stationary phase (changing polarity)
HOW TO IMPROVE RESOLUTION IN HPLC

ISOCRATIC VS. GRADIENT ANALYSIS
Gradient analysis is preferred for more complex samples containing analytes of diverse
polarities.
Isocratic analysis : the same mobile phase is used throughout the elution of the
entire sample. Isocratic analysis is good for simple mixtures,
Gradient analysis : the strength of the mobile phase is increased with time during
sample elution

ISOCRATIC ANALYSIS
GRADIENT ANALYSIS

• Higher peak capacity (fit more peaks in the chromatogram)
Advantages of gradient analysis are:
• Better for complex samples and applications that require quantitation of all peaks or
multiple analytes of diverse polarities
• Better resolution of early and late eluting peaks
• Better sensitivity of late eluting peaks
• Typically longer assay times since column must be equilibrated with the initial mobile phase
Disadvantages are:
• More complex HPLC instrument is required (i.e., binary pump)
• Method development, implementation, and transfer are more difficult

The chromatogram is useful for both qualitative and quantitative analysis.
a chromatogram is plot the signal of detector that responds to solute concentration
as function of time, and a series of peaks is obtained
A chromatogram provides only a single piece of qualitative information about
each species in a sample, namely, its retention time.
It is a widely used tool for recognizing the presence or absence of components of mixtures

Quantitative column chromatography is based upon a comparison of either the
height or the area of the analyte peak with that of one or more standards.
If conditions are properly controlled, these parameters vary linearly with concentration.
Most modern chromatographic instruments are equipped with digital electronic
integrators that permit precise estimation of peak areas.
Peak areas are a more satisfactory analytical variable than peak heights.

•The most straightforward method for quantitative chromatographic analyses involves
the preparation of a series of standard solutions that approximate the composition of the
unknown.
Chromatograms for the standards are then
obtained and peak heights or areas are plotted
as a function of concentration.
A plot of the data should yield a straight line
passing through the origin.

2.9 EFFECTS OF TEMPERATURE IN HPLC19
It is not possible to set generally valid rules about the influence of
temperature on HPLC separations. At increased temperature the
performance of a column often increases because of the decrease of
mobile phase viscosity which improves mass transfer; however, it is
also possible that performance decreases.
The separation factor can increase or decrease. An advantage is the
shortening of analysis time due to the possibility to use higher flow-
rates of the mobile phase due to the increase in diffusion coefficients. If
the eluent or the sample solution are viscous it is even necessary to
work at higher temperatures: less pressure is needed to pump the
mobile phase or it is possible only under these circumstances to inject
the sample, respectively.

Asymmetry Factor As
The ASTM International standards organization recommends
calculating column symmetry or asymmetry (As) as the back-to-front
ratio of a bisected peak measured at 10% of height
A tailing peak has a front of greater than 1.0, while a fronting peak has a front of
less than 1.0. The U.S. Pharmacopeia (USP) has also recommended measuring
tailing factor (T) as the back-to-front ratio of a bisected peak measured at 5% of
height. The ratio is made by dividing the total width by twice the front width.
USP tailing factor T

HPLC - Resolution
a slight partial
separation
partial separation
the minimum separation
required
baseline separation

Under ideal conditions, chromatographic peaks should have Gaussian peak
shapes with perfect symmetry. In reality, most peaks are not perfectly
symmetrical and can be either fronting or tailing

Indicate which of the following parameters can decrease or increase
column efficiency in liquid chromatography.
• Very low flow rate
• Large particle size of stationary phase
• Small particle size of stationary phase
• Thick stationary-phase coating
• Thin stationary-phase coating
• Regularly shaped particles of stationary phase
• Irregularly shaped particles of stationary phase
• High temperature
• Low temperature
• Uneven stationary-phase coating
• Even stationary-phase coating
• Uniform stationary-phase particle size
• Non-uniform stationary-phase particle size
• Low diffusion coefficient in the mobile phase
• High diffusion coefficient in the mobile phase
• Low diffusion coefficient in the stationary phase
• High diffusion coefficient in the stationary phase.

The most effective and convenient way to alter the retention factor
of a peak is to adjust the ‘solvent strength’ of the chromatographic
mobile phase.

Separation Factor (  ) or Selectivity Factor
is a measure of relative retention k2/k1 of two sample components
A chromatogram of two peaks with a selectivity factor (α) of 1.3.
Selectivity must be >1.0 for peak separation.

Buffers
In RPLC, the ionized form of the solute does not partition
well into the hydrophobic stationary phase and has lower k
than the neutral form.
Figure 2.13 shows the retention map of two basic drugs.

Figure 2.13. Retention map and chromatograms of two
basic antidepressants using mobile phases at various pH

Note that at pH 2.0, both ionized solutes are not
retained and elute as a single peak.
At pH 8, the solutes are partially ionized and separate
well.
At pH 10, both un-ionized solutes are highly retained
and resolved.
Buffers are required to control the pH of the mobile
phase

Injector
Detector
Column
Solvents
Mixer
Pumps
Waste
mobile phase
stationary phase
( eluent )

86
Injector
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Solvents
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Chromatogram
Start Injection
mAU
time
High Performance Liquid Chromatograph
Separation Process and Chromatogram for Column Chromatography

87
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4. Fields of used of HPLC
Biogenic substances
Sugars, lipids, nucleic acids,
amino acids, proteins,
peptides, steroids, amines, etc.
Drugs, antibiotics, etc
Medical products :
Vitamins, food additives, sugars,
organic acids, amino acids, etc.
Food products
Inorganic ions
Hazardous organic substances, etc.
Environmental samples
Synthetic polymers,
additives, surfactants, etc.
Organic industrial products

105
HPLC Applications
Chemical
Environmental
Pharmaceuticals
Consumer Products
Clinical
polystyrenes
dyes
phthalates
tetracyclines
corticosteroids
antidepressants
barbiturates
amino acids
vitamins
homocysteine
Bioscience
proteins
peptides
nucleotides
lipids
antioxidants
sugars
polyaromatic hydrocarbons
Inorganic ions
herbicides

UV Spectrum
Chromatogram
Reset
Ready
UV Spectrum
{shows full UV abs.}
Chromatogram
{shows peaks, Rt}
ABS.
Time
ABS.
Wavelength
UVmax
UVmax
Rt Rt

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