Ravi Pratap Pulla: Asso - Professor, SSJ College of Pharmacy, V.N.Pally, Gandipet, Hyderabad-75

HPLC
I/II, Ist Semester M.Pharmacy Dept . Of Pharmaceutical Analysis, JNTUH Lecture by:
RAVI PRATAP PULLA
M.Pharm., Ph.D
Asso.Professor, SSJ College of Pharmacy, V.N.Pally, Gandipet, Hyderabad-75.

SSJCP, Department of Pharmaceutical Analysis
HPLC THE DEVELOPMENT OF A NAME

PERFORMANCE PRESSURE Price Prestige Peak
Profit
High Propaganda Promise Philosophy Polite Liquid
Chromatography
Problem
Ph (F) antasy Pragmatic Pleasure Passion
Introduction to Liquid Chromatography

Columns System Components Applications Troubleshooting
A Brief History of Chromatography

1903: Russian botanist Mikhail Tswett separated plant pigments 1938: Russian scientists Izmailov and Shraiber use drop chromatography. Later perfected as Thin Layer Chromatography (TLC) by Kirchner in the U.S. 1952: Martin and Synge receive Nobel Prize for invention of partition chromatography or plate theory to describe column efficiency.
1966: HPLC was first named by Horvath at Yale University but HPLC didnt catch on until the 1970s 1978: W.C. Stills introduced flash chromatography, where solvent is forced through a packed column with positive pressure.
Modern HPLC
Late 1970s/early 1980s
Instrumentation developed for high pressure solvent delivery: pumps, autosamplers, diode array detectors More uniform packing material produced columns for
Last 20 years
Nothing really new, but by returning to the basic theory of chromatography, even better columns are on the market: smaller particle sizes which yield faster separations, but require hardware to withstand higher pressures.
What is Chromatography?
Separation of a mixture into individual components. The separation uses a Column (stationary phase) and Solvent (mobile phase). The components are separated from each other based on differences in affinity for the mobile or stationary phase. The goal of the separation is to have the best RESOLUTION possible between components.
CHROMATOGRAPHY IS INCOMPLETE WITHOUT LEARNING FEW BASIC TERMINOLOGIES

For any further clarification or details of the below content(s) feel free to mail me :
ravipratappulla@gmail.com
Absorption
Additive Adsorbent Adsorption Adsorption isotherm Affinity chromatography
Bonded phase
Breakthrough volume Capillary column Capillary LC Cartridge column Cation exchange chromatography
Agarose Alumina
Amphoteric ion-exchange phase Analyte Anion exchange chromatography Bed volume
Channeling Chemisorption
Chiral stationary phase Chlorosilane Co-ion Column back pressure
BET (Brunauer, Emmet & Teller) Column chromatography method SSJCP, Department of Pharmaceutical Analysis
Column plate number
Eluite
Column switching Column volume Competing base Counterion Coverage Cross-links Degassing
Dead time (to / tm ) Displacement chromatography Dynamic coating
Effluent Eluate
Eluent
Elute Elution Exclusion chromatography (Size) Extra column effects Fast protein LC (FPLC) Frontal chromatography Gel filtration chromatography (GFC) Gradient elution Graphitized carbon packing Guard column Heart cutting
Hold-up volume ( VM or tM )
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Hydrophobic interaction chromatography (HIC) Ion chromatography
Ion exclusion Ion moderated partioning chromatography (IMPC)
Imprinted phases
Indirect detection Injector (sample)
Ion pair chromatography (IPC)

Linear chromatography Linear velocity
Inlet
In-line filter Interparticle porosity (ee)
Liquid chromatography
Mobile phase velocity Open tubular column
Interstitial volume
Intraparticle porosity (ei) Intraparticle volume
Partition chromatography
Packed column Peak
Ion exchange chromatography

Ion chromatography (IC)
Peak area
Peak maximum
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Peak width Phase ratio
Retention factor (k) Retention volume (VR or tR)
Plate height (H)

Plate number (N)
Separation factor (a)

Solid support
Pressure drop
Resolution (Peak) [ Rs ]/ Resolution(R)
Solute
Reduced mobile phase velocity (n) Stationary phase Tailing
Reduced plate height (h)

Relative Retention time (RRT)
Void volume
Retention time (tR )
Interparticle time (tZ)

Selectivity factor ()
Capacity factor (k)

Dead Volume(Vd)
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Activity Asymmetry
Adsorption chromatography Back pressure
Back flushing
Baseline Baseline resolved peak
Band spacing
Baseline noise Breakthrough volume
Buffer
Capacity factor Channeling
Calibration standard
Chain length Chromatogram
Chromatographic conditions
Chromatographic system Dead volume (Vm)
Chromatographic resolution
Column performance Dead time (tm)
Detection
Detection threshold
Detector
Detector linearity
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Detector sensitivity Electrochemical detector
Differential Refractive Index( RI) Elution order
Elution chromatography
Elution volume External standard
Eluotropic sequence
Extra column volume Flow rate
Fluorescence detector
Fronting Hydrophilic
Frit
HETP Hydrophobic
Internal standard
Interstitial particle volume Ion suppression
Integrator
Ion exchanger Isocratic analysis
Isothermal chromatography
Loading
Ligand
matrix
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Organic modifier Partially resolved peaks
Overload Particle size (medium)
Particle size distribution

Peak area Peak height
Peak broadening
Peak base Peak identification
Peak Quantitation
Phase system Pore diameter
Peak shape
Polarity Pore volume
Post column derivatization

Pulsating flow Regeneration
Pre column
Recycling Retention
Retention time
Sample
Retention volume
Sample capacity
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Sample preparation silanization Sorbent S.P Specific surface Vacancy chromatogram Void time
Separation capacity Silanol groups S.P chemically bonded Surface modification SFC( supercritical fluid chromatography) Void
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IUPAC RECOMMENDATIONS & FREQUENTLY USED SYMBOLS IN PARAMETER SYMBOL CHROMATOGRAPHY Separation factor
Selectivity factor (up to 1993 A.D) Area Diameter a/A de
Diffusion coefficient
Porosity Flow rate (volumetric) Plate height Viscosity Equilibrium distribution constant Rate constant Retention factor
d
/ t f h k k k
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PARAMETER Capacity factor Length of the column Plate number /number of theoretical plates Density
SYMBOL k l / L n / N
Pressure
Pressure (relative) Radius Temperature (absolute) Time Retention time Velocity (linear) Volume
p / P
p r t /T t tr / tR u v
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PARAMETER Retention volume Mass (Weight) Peak width Difference Partial diameter Flow Height equivalent of a theoretical plate(HETP)
SYMBOL vr w w
dp F H
Internal diameter of the column

Wavelength Iso electric point Resolution Death time
I.D
pKa R tm / t0
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PARAMETER Gradient time Net retention time Linear velocity Dead volume of apparatus Pore volume
SYMBOL tG tR' Vd Vp
For any further clarification or details of the above content(s) feel free to mail me :
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The Most Basic Explanation of Chromatography Ever
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Drugs in multi component dosage forms, analyzed by HPLC method because of the several advantages like:
Improved resolution of the separated substances
Faster separation times

The improved accuracy, precision, & sensitivity with which the separated substances may be quantified.
How Do You Get Separation? Hardware: pumps, injector, detector
Column: particle diameter, column size, packing materials

Our seminar will focus on the contribution of each factor to perform separations.
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Column Considerations Theory (including, well...you know) Different Stationary Phases
Hardware Components Pumps, Injectors, Detectors, etc. Examples of Application-Specific Configurations

Applications Pharmaceuticals and Proteomics Food and Beverage, Environmental Research and Method Development
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System Troubleshooting Leaks, Reproducibility, Column Care, and More Chromatography Software Method and Sequence Setup Calibration Curves and Reporting Chromatography Hardware Modular LC-20 Prominence Integrated LC-2010HT, Empower 2
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Modern HPLC v/s Traditional LC Methods

Classical open-column LC. Thin-Layer Chromatography (TLC) and paper chromatography. In modern HPLC the columns and packings are, in general, highly refined, high in resolving capacity, and are reusable.
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HPLC and Pre-HPLC Techniques
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MODES OF SEPARATION IN HPLC

There are different modes of separation in HPLC:
Normal phase mode Reversed phase mode RP - Ion pair chromatography Affinity/Bioaffinity chromatography
Size exclusion chromatography

Displacement chromatography
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Based on mode of chromatography
Normal phase mode

Reverse phase mode
Based on principle of separation

Adsorption chromatography Ion exchange chromatography Ion pair chromatography Size exclusion chromatography Affinity chromatography
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Based on elution technique
Isocratic separation
Gradient separation
Based on the scale of operation

Analytical HPLC Preparative HPLC Based on the type of analysis Qualitative analysis Quantitative analysis
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COLUMN TYPES Normal Phase LC

Polar - stationary phase: Silica
Nonpolar - mobile phase: Hexane, Ethyl acetate

The LEAST polar compound comes out first Generally used for separation of non polar compounds.
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Normal Phase HPLC Columns

Cyano : `Rugged, moderate polarity, general use More polar and retentive
Highly polar, less stable Very rugged, low cost, adsorbent & Unbonded
-OH (Diol)
Amino Silica
:
: :
NOTE: The cyano column with a low polarity mobile phase (hydrocarbon with a small amount of another solvent) will act as a normal phase column.
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this method separates analytes based on their affinity for a polar stationary surface such as silica based on analyte ability to engage in polar interactions (such as hydrogen-bonding or dipole-dipole type of interactions) with the sorbent surface. Adsorption strengths increase with increased analyte polarity interaction strength depends on the functional groups present in the structure of the analyte molecule, but also on steric factors
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more polar solvents in the mobile phase will decrease the retention time of analytes hydrophobic solvents tend to induce slower elution (increased retention times) traces of water in the mobile phase tend to adsorb to the solid surface of the stationary phase forming a stationary bound (water) layer which is considered to play an active role in retention. governed mechanism almost exclusively by an adsorptive
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Reversed-Phase LC
Nonpolar - stationary phase: C8, C18 Polar - mobile phase: Water, ACN, Methanol The MOST polar compound comes out first Generally used for separation of polar compounds
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RP-HPLC Columns
C18, C8
C3, C4
:
:
Rugged, general purpose, highly retentive

Less retentive, used mostly for peptides & proteins Greater selectivity than alkyl-bonded
Phenyl
Cyano
Amino
NOTE
:
:
:
Moderate retention, normal & rev. phase

Weak retention, good for carbohydrates
The cyano column with a high polarity mobile phase (Water/MeOH) will act as a RP- Column.
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stationary phase is a silica which has been surfacemodified with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. retention time is longer for molecules which are less polar, while polar molecules elute more readily can increase retention times by adding more water to the mobile phase the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase
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decrease retention time by adding more organic solvent to the eluent RP-HPLC operates on the principle of hydrophobic interactions RP-HPLC allows the measurement of these interactive forces. The binding of the analyte to the stationary phase is proportional to the contact surface area around the nonpolar segment of the analyte molecule upon association with the ligand on the stationary phase.
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solvophobic effect is dominated by the force of water for
"cavity-reduction" around the analyte and the C18-chain versus the complex of both.
The retention can be decreased by adding a less polar solvent
(methanol, acetonitrile) into the mobile phase to reduce the surface tension of water.
Gradient elution uses this effect by automatically reducing
the polarity and the surface tension of the aqueous mobile phase during the course of the analysis.
Structural properties of the analyte molecule play an
important role in its retention characteristics.

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an analyte with a larger hydrophobic surface area (C-H, C-C, and generally non-polar atomic bonds, such as S-S and others) is retained longer because it is non-interacting with the water structure. analytes with higher polar surface area (conferred by the presence of polar groups, such as -OH, -NH2, COO or -NH3+ in their structure) are less retained as they are better integrated into water. interactions are subject to steric effects in that very large molecules may have only restricted access to the pores of the stationary phase, where the interactions with surface ligands (alkyl chains) take place.
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surface hindrance typically results in less retention. Retention time increases with hydrophobic (non-polar) surface area. Branched chain compounds elute more rapidly than their corresponding linear isomers because the overall surface area is decreased. organic compounds with single C-C-bonds elute later than those with a C=C or C-C-triple bond, as the double or triple bond is shorter than a single C-C-bond.
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mobile phase surface tension (organizational strength in eluent structure), other mobile phase modifiers can affect analyte retention.
entropy of the analyte-solvent interface is controlled by surface tension, the addition of salts tend to increase the retention time.
mobile phase pH can change the hydrophobic character of the analyte. For this reason most methods use a buffering agent, such as sodium phosphate, to control the pH.
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Ammonium formate is commonly added in mass spectrometry to improve detection of certain analytes by the formation of analyte-ammonium adducts. volatile organic acid such as acetic acid, or formic acid, is often added to the mobile phase if mass spectrometry is used to analyze the column effluent. Trifluoroacetic acid is used infrequently in mass spectrometry applications due to its persistence in the detector and solvent delivery system, but can be effective in improving retention of analytes such as carboxylic acids in applications utilizing other detectors, as it is a fairly strong organic acid.
43
Reversed phase columns consist of alkyl derivatized silica
particles and should never be used with aqueous bases as these will destroy the underlying silica particle.
Can be used with aqueous acid, but the column should not
be exposed to the acid for too long, as it can corrode the metal parts of the HPLC equipment.
A good test for the metal content of a column is to inject a
sample which is a mixture of 2,2'- and 4,4'- bipyridine.

Because the 2,2'-bipy can chelate the metal, the shape of
the peak for the 2,2'-bipy will be distorted (tailed) when metal ions are present on the surface of the silica.
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TYPICAL COLUMN SIZES
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Particle size: 5 m, 3 m, and smaller Mono dispersed means particles are the same size Very important for stable pressure and flow Smaller particles produce higher system pressure Pore size: 100-120 A is typical Surface area: 300-350 m2/g Carbon load: 9-12% for C8, 16-20% for C18 Higher carbon load = better resolution but longer run times Lower carbon load = shorter run times, but may change selectivity v/s higher carbon load SSJCP, Department of Pharmaceutical Analysis
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RP-HPLC MECHANISM
Synthesis of RP Packing RP Column Properties RP Retention Mechanisms Important RP parameters RP Optimization
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Synthesis of RP Packing
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RP COLUMN PREPARATION
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COMMON RP PACKING
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RP COLUMN PROPERTIES
Hydrophobic Surface Particle Size and Shape Particle Size Distribution Porosity, Pore Size and Surface Area
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PARTICLE SIZE
Columns have a distribution of particle sizes
Reported particle diameter is an average
Broader distribution ---> broader peaks

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Particle Size Distribution of several column batches
Copyrights: Neue, HPLC Columns Theory, Technology and Practice, Wiley, 1997, p.82
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RP MECHANISM (SIMPLE)
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RP Mechanism (Advanced)
Classical measures of retention capacity factors partition coefficients Vant Hoff Plots Give bulk properties only do not give molecular view of separation process
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PROPOSED RP MECHANISMS
Hydrophobic Theory Partition Theory Adsorption Theory
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Chromatography of cavities in solvent created by hydrophobic portion of analyte molecule Surface Tension Interaction of polar functions with solvent Stationary phase is passive
HYDROPHOBIC THEORY
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PARTITION THEORY Analyte distributes between aqueous mobile

phase and organic stationary phase
Correlation between log P and retention organic phase is attached on one end Does not explain shape selectivity effects
59
Analytes land on surface - do not penetrate
ADSORPTION THEORY
Non-polar interactions between analyte hydrophobic portion and bonded phase Weak interactions dipole-dipole dipole-induced dipole induced dipole-induced dipole
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None of the above can completely explain all of the observed retention in RP-HPLC
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IMPORTANT REVERSED PHASE PARAMETERS
Solvent (mobile phase ) Strength Choice of Solvent Mobile Phase pH Silanol Activity
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SOLVENT STRENGTH
Water is weak solvent
Increased organic ---> decreased retention Organic must be miscible with water
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EFFECT OF SOLVENT
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SOLVENT STRENGTH
COPYRIGHTS:Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 286.
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VARYING SELECTIVITY
30% MeCN 70% Water 45% MeOH 55% Water
30x0.46 cm C-18, 1.5 mL.min,254 nm, 10 mg each
COPYRIGHTS:Snyder and Kirkland, introduction to Modern Liquid Chromatography, Wiley, 1979, p. 287.
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pH
Affects ionizable compounds organic acids organic bases In reversed phase we need to suppress ionization as much as possible May need very precise pH control
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pH Effect on Retention
1. Salicylic acid 2. Phenobarbitone 3. Phenacetin 4. Nicotine
5. Methylampohetamine
30x0.4 cm C-18, 10 mm, 2 mL/min, UV 220 nm
COPYRIGHTS: Snyder and Kirkland, Introduction to Modern Liquid Chromatography, Wiley, 1979, p. 288.
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Use of Buffers
0.1 pH unit ---> significant effect on retention Buffer mobile phase for pH reproducibility
pH of buffer should be within 1 pH unit of pKa of acid (best at pH = pKa) Buffers weak (100 mM or less)
Check solubility
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Common buffers
Buffer pKa Values Phosphate 2, 7
Acetate
4.75
Citrate
3.08, 4.77, 6.40
Useful buffering between pH 2-8.
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Silanol Activity
RP ligands occupy about 50% of silanols Others are active Weak acids
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Silica Surface
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Dealing with Residual Silanols

Silanols cause peak tailing and excessive retention
Endcapping bond a smaller group (helps a little) Pre-treatment of silica fully hydroxylated best high purity best
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Silanol Interactions
Hydrogen bonding Dipole-dipole Ion exchange Low pH --> silanols protonated Add basic modifier (TEA) to compete for sties
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pH Effect on Tailing
Neue, p196
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RP Optimization
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IDEALIZED HPLC SEPARATION
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VOID VOLUME The void volume is the amount of dead volume in the column that is not taken up by the particles of stationary phase. In general, there is approximately 0.1 mL of void volume for each cm of column length, for columns with a 4.6 mm i.d. and 5 m particles
Vm 0.5dc2L
Where,
Vm is the column volume in mL, L is the column length in cm, and dc is the inner diameter in cm
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The void volume is exactly determined by injecting a compound that is completely unretained, then using the chromatogram to calculate void volume. void volume = Elution time x flow rate
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FACTORS INFLUENCING RESOLUTION

Capacity Factor, k
Selectivity Factor, Efficiency, N
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RESOLUTION
For closely eluting or adjacent peaks, the resolution equation may be expressed as:
Rs 1 / 4[( 1) / ] N [k ' /(1 k ' )]

The terms of capacity factor (k), selectivity (), and efficiency (N) all contribute to resolution
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THE RESOLUTION EQUATION

Resolution is defined as the completeness of separation from one analyte to another In general, resolution may be expressed as: Rs = 2(Vrb - Vra)/(Wa + Wb) = 2(trb - tra)/ (Wa + Wb)
Where, Vra/b = retention volume of peak a/b tra/b = retention time of peak a/b SSJCP, Department of peak Pharmaceutical Analysis W /b = width of a/b
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CAPACITY FACTOR, k
The relative degree to which an analyte component is = (Vrthrough - V0)/V = (t 0 given r - t0)/t 0 delayed as itk is eluted a system (retentivity).
Where,
Vr = peak retention volume V0 = column void volume tr = peak retention time t0 = peak void time
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EFFECT OF k ON OVERALL RESOLUTION

As k grows larger, its effect reaches a limit at a value of about 10.
Since k depends on retention time, longer columns eventually have a diminished effect on resolution.
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INFLUENCING THE CAPACITY FACTOR (k)

Retentivity (k) decreases 2 - 3 fold for each 10% increase in mobile phase strength. Which of these is easiest to change?? Mobile Phase Strength As per the rule of thumb, altering the mobile phase strength also alters the retention of the analytes. Bonded Phase Functionality (RP) As the bonded phase hydrophobicity increases (increasing alkyl chain length, etc.) so will the retention of the analytes.
Temperature As temperature increases, the retention time decreases. This does not necessarily result in poorer separation because of the other factors in the resolution equation.
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Mobile Phase Strength v/s

0.079
100% 100%
4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water
90% 90%
0.212
Capacity Factor for Butyl Paraben (Peak 4)
0.472
80% 80%
70% 70%
1.127 2.813
60% 60%
7.666
50% 50%
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Temperature Effect on
50C
2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water
45C
40C
35C
30C
25C
20C
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Summary of
Effects
A larger value of k means better resolution...to a certain extent (k = 10 maximum) Increasing the mobile phase strength decreases k Increasing the temperature decreases k, but may not result in a bad separation based on the other factors affecting resolution.
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Selectivity Factor,
The selectivity or separation factor represents the ratio of any two
adjacent k values, there by describing the relative separation of adjacent peaks.
This relationship is expressed as:

= kb/ka
If = 1, two components are perfectly overlapping For early eluting peaks you want to be large for good resolution. For later eluting peaks, can be smaller and still have acceptable
separation.
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Effect of on Overall Resolution

Remember the resolution equation?
Rs 1 / 4[( 1) / ] N [k ' /(1 k ' )]

Lets only look at the part involving
Rs 1 / 4[( 1) / ]
And see how much resolution will improve with small changes in
91
For an value of 1.1, the contribution of the selectivity term is (1.1 1) / 1.1 = 0.09 For an value of 1.4, the contribution of the selectivity term is (1.4 1) / 1.4 = 0.29 So, a very small change in leads to a more than THREE-FOLD increase in the contribution to resolution.
92
As grows larger, its effect reaches a limit at a value of about 5. Since depends on components retention factor k, longer columns eventually have a diminished effect on resolution.
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Influencing the Selectivity Factor

Which of these is easiest to change?? Mobile Phase Type The importance of the type of interactions between the mobile phase and analytes is critical to the optimization of the selectivity of a system. Column Type -
The bonded phase functionality can be selected by its chemical nature to provide better selectivity in an analytical method.
Temperature Selective interactions between analyte molecules and the stationary phase may not become evident until a critical temperature is attained. SSJCP, Department of Pharmaceutical Analysis
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Summary of Effects
Since is the ratio of two k values, the same general
statements apply:
Increasing the mobile phase strength decreases individual
values of k, but their ratio () may affect resolution
Increasing the temperature decreases individual values of
k, but their ratio () may significantly affect resolution.
A small increase in leads to a large increase in resolution
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Column Efficiency, N
The column efficiency is defined as the degree to which a column and/or other system components can physically and chemically affect the separation of analytes. As column efficiency increases, analyte components will elute in a smaller volume of the mobile phase, usually observed as narrower or sharper peak shapes. Column efficiency is generally expressed in terms of theoretical plate number.
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Calculation of Theoretical Plates
N = A(tr /W)2 A Method Width measured at Wi 4 Inflection point (60.7% of peak height) Wh 5.54 Height 50% of peak height W3s 9 3s 32.4% of peak height W4s 16 4s 13.4% of peak height W5s 25 5s 4.4% of peak height Wb 16 Tangent Baseline, following tangent drawing Constants A are different at each peak width, assuming a perfect Gaussian shape. Real-world peaks often have tailing, so widths measured at the lower part of the peak more accurately reflect the tailing when calculating N. W
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Calculation of Efficiency, N
Width measured at the baseline after tangent lines are drawn on the peak. Used when tailing is minimal.
Width measured at 4.4% of peak height, no tangents drawn. Used when tailing is significant.
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Effect of N on Overall Resolution

Do you STILL remember the resolution equation?
Rs 1 / 4[( 1) / ] N [k ' /(1 k ' )]

Now lets look at the part involving N
Rs 1 / 4 N
And see how much resolution will improve with changes in N
99
Since the contribution of N to resolution is a square root, doubling N from 5000 to 10,000 only increases the contribution to resolution by 41%. To double the effect on resolution coming from N, we have to increase the value of N by a factor of 4
PLATE
Plates 5,000 5000 10,000 10,000 20,000

20,000
N 70.7 70.7 100 100 141.4

141.4
CONTRIBUTIO N Contribution
- - - - ----41% 41% 100%

100%
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Effect of N on Overall Resolution
Note that there is no flattening of the curve like with k and . Resolution will continue to increase as theoretical plates increase. SSJCP, Department of Pharmaceutical Analysis
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Influencing the Efficiency, N

Particle Size and Size Distribution The smaller the particle size and the narrower the range of the particle size distribution, the more efficient the column.
Packing Type Totally porous particles will also have greater efficiency than solid or pellicular-shaped packing's, due to the additional surface area attributable to the pores.
Mobile Phase Viscosity As mobile phase viscosity increases, molecular movement through the mobile phase is inhibited.
Temperature For reverse phase chromatography, an increase in efficiency, N, may be realized as column temperature is increased. SSJCP, Department of Pharmaceutical Analysis
10
Effect of Particle Size on N

Smaller particle sizes result in higher numbers of theoretical plates
Column Diameter (mm) 10 4.6 4.6 4.6 4.6 4.6 3 2 2 2 2 1 1 1 1 1 Column Length (cm) 25 25 25 10 10 3 10 25 25 10 10 25 25 25 10 10 Particle Size (m) 10 10 5 5 3 3 5 10 5 5 3 10 5 3 5 3 4 Peak Width (L) 1118 237 167 106 82 45 45 45 32 20 15 11 8 6 5 4 Theoretical Plates per centimeter 333 333 667 667 1111 1111 667 333 667 667 1111 333 667 1111 667 1111
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Relative Influence of All Factors on Resolution

Paramete r Change Standard +10% N -25% N -50% N -60% N -75% N +10% k +10% N 10,000 11,000 7,500 5,000 4,000 2,500 10,000 10,000 k 2 2 2 2 2 2 2.2 2 1.1 1.1 1.1 1.1 1.1 1.1 1.1 1.2 Rs 1.52 1.59 1.31 1.07 0.96 0.76 1.56 2.78
Note that changing a very small amount has the biggest effect
104
Review of Factors
PARAMTER INFLUENCED BY Column, system flow path, configuration MP strength M.P & S.P type All of the above TARGET VALUE Minimum of 400 theoretical plates /cm 1.0 - 10 1.1 - 2 1.3 1.5 or greater
Efficiency, N
Capacity factor, k Selectivity, Resolution, Rs
105
Normal Phase v/s Reversed Phase

PARAMETER
Polarity of packing
Polarity of solvent
NP
Medium to high Low to medium
RP
Low to medium Medium to high
Elution sequence Low polarity first High polarity first Increase solvent polarity
Faster elution
Slower elution
10
ION EXCHANGE CHROMATOGRAPHY
107
based on the attraction between solute ions and charged sites bound to the stationary phase. The stationary phase contains ionic groups like NR, SO which interact with the ionic groups of the sample molecules. This method is suitable for the separation of charged molecules only.
Solute ions of the same charge as the charged sites on the column are excluded from binding
solute ions of the opposite charge of the charged sites of the column are retained on the column. Strong acids & basic compounds may be separated by RP mode by forming ion pairs with suitable counter ions. SSJCP, Department of Pharmaceutical Analysis
108
Solute ions that are retained on the column can be eluted from the column by changing the solvent conditions They include: increasing the ion effect of the solvent system
by increasing the salt concentration of the solution increasing the column temperature
changing the pH of the solvent
109
ion exchangers favor the binding of ions of higher charge and smaller radius. increase in counter ion (with respect to the functional groups in resins) concentration reduces the retention time. decrease in pH reduces the retention time in cation exchange while an increase in pH reduces the retention time in anion exchange. lowering the pH of the solvent in a cation exchange column, more hydrogen ions are available to compete for positions on the anionic stationary phase, thereby eluting weakly bound cations.
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TYPES OF ION EXCHANGERS

Polystyrene resins These allow cross linkage which increases the stability of the chain. Higher cross linkage reduces swerving, which increases the equilibration time and ultimately improves selectivity. Cellulose and dextran ion exchangers (gels) These possess larger pore sizes and low charge densities making them suitable for protein separation. Controlled-pore glass or porous silica
111
Examples
Stationary phase contains charged groups SAX (Strong Anion Exchange): NH3+ WAX (Weak Anion Exchange): NR2H+(DEAE)
[Di Ethyl Amino Ethanol] SCX (Strong Cation Exchange): SO3 WCX (Weak Cation Exchange): CarboxyMethyl (CM) More highly charged analytes have stronger retention More bulky stationary phases have weaker retention
112
IEC is widely used in the following applications: water purification preconcentration of trace components ligand-exchange chromatography ion-exchange chromatography of proteins high-pH anion-exchange chromatography of carbohydrates and oligosaccharides
113
AFFINITY/ BIOAFFINITY CHROMATOGRAPHY
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AFFINITY CHROMATOGRAPHY
It uses highly specific biochemical interactions for separations. The stationary phase contains specific groups of molecules which can absorb the sample if certain steric & charge related conditions are satisfied. This technique can be used to isolate proteins, enzymes, receptors , ligands as well as antibodies from complex mixture.
11
Affinity chromatography can be used to: Purify and concentrate a substance from a mixture into a buffering solution Reduce the amount of a substance in a mixture
Discern what biological compounds bind to a particular substance Purify and concentrate an enzyme solution.
116
Size Exclusion LC (or) Gel Permeation (or) Gel filtration
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Stationary phase is a polymer (polystyrene-divinyl benzene or acrylamide) with a defined pore size Large compounds cannot fit into the pores and elute first Used to determine molecular weight distribution of polymers Separates molecules according to their molecular mass. Largest molecules are eluted first and smaller molecules last. useful for determining the tertiary structure andquaternary structure of purified proteins. used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. widely used for the molecular weight determination of polysaccharides.
118
larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time. separates particles on the basis of molecular size (actually by a particle's Stokes radius or Stokes-Einstein radius, or hydrodynamic radius (RH). named after George Gabriel Stokes is the radius of a hard sphere that diffuses at the same rate as the molecule. generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of the purification. SSJCP, Department of Pharmaceutical Analysis
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The main application of gel-filtration chromatography: fractionation of proteins and other water-soluble polymers while gel permeation chromatography is used to analyze the molecular weight distribution of organicsoluble polymers. Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges.
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DISPLACEMENT CHROMATOGRAPHY
A molecule with a high affinity for the chromatography matrix (the displacer) will compete effectively for binding sites, and thus displace all molecules with lesser affinities displacement chromatography has advantages over elution chromatography in that components are resolved into consecutive zones of pure substances rather than peaks. because the process takes advantage of the nonlinearity of the isotherms, a larger column feed can be separated on a given column with the purified components recovered at significantly higher concentration.
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Aqueous Normal-Phase Chromatography (ANP)

ANP is a chromatographic technique which encompasses the mobile phase region between RPC and organic normal phase chromatography (ONPC). This technique is used to achieve unique selectivity for hydrophilic compounds, showing normal phase elution using reversed-phase solvents.
122
ISOCRATIC & GRADIENT ELUTION

A separation in which the mobile phase composition remains constant throughout the procedure is termed isocratic (constant composition).
Word was coined by Csaba Horvath A separation in which the mobile phase composition is changed during the separation process is described as a gradient elution In isocratic elution, peak width increases with retention time linearly leads to the disadvantage that late-eluting peaks get very flat and broad. SSJCP, Department of Pharmaceutical Analysis
12
Gradient elution decreases the retention of the latereluting components so that they elute faster, giving narrower (and taller) peaks for most components improves the peak shape for tailed peaks, as the increasing concentration of the organic eluent pushes the tailing part of a peak forward. increases the peak height (the peak looks "sharper") may include sudden "step" increases in the percentage of the organic component, or different slopes at different times.
124
In isocratic elution, the selectivity does not change if the column dimensions (length and inner diameter) change In gradient elution, the elution order may change as the dimensions or flow rate change The driving force in RPC originates in the high order of the water structure. The role of the organic component of the mobile phase is to reduce this high order and thus reduce the retarding strength of the aqueous component.
125
ISOCRATIC SYSTEM
Same mobile phase concentration throughout the separation Use 1 pump and pre-mix solvents Use 1 pump and a valve for 4 different solvents
Use 2 pumps and vary the amount coming from each pump
126
ISOCRATIC SEPARATION
1 pump and premixing
4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water
127
1 pump with valve and premixing
To Column
A = 80% Methanol, 20% Water B = 70% Methanol, 30% Water C = 60% Methanol, 40% Water
ABCD
D = 50% Methanol, 50% Water
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1 pump with mixer let the pump do the work!
To Column
Method 1: A.CONC = 20%, B.CONC = 80% Method 2: A.CONC = 30%, B.CONC = 70% Method 3: A.CONC = 40%, B.CONC = 60%
ABCD
Method 4: A.CONC = 50%, B.CONC = 50%
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LOW PRESSURE GRADIENT

1 Pump, solvents are mixed before the pump Requires degassing
To Column
ABCD
13
HIGH PRESSURE GRADIENT Ternary Gradient

Binary Gradient 2 Pumps and Mixer
. . .
3 Pumps and Mixer

. . .
. . . . . .
. . .
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HIGH v/s LOW PRESSURE GRADIENT

High Pressure Gradient
Multiple pumps are used with a mixer after the pumps
Low Pressure Gradient

Solvents are mixed before the pump
13
Gradient v/s Isocratic Conditions: Summarized

Isocratic mobile phase solvent composition remains constant with time Best for simple separations Often used in quality control applications that support and are in close proximity to a manufacturing process Gradient mobile phase solvent (B) composition increases with time Best for the analysis of complex samples Often used in method development for unknown mixtures Linear gradients are most popular (for example, the gradient shown at right) SSJCP, Department of Pharmaceutical Analysis
13
PRINCIPLE OF SEPARATION
The
principle of separation is Adsorption.
Separation of components takes place because of the difference in affinity of compounds towards stationary phase.
13
The principle of separation in normal phase mode and reverse phase mode is adsorption. The component which has more affinity towards the adsorbent, travels slower. The component which has less affinity towards the stationary phase travels faster.
Since no two components have the same affinity towards the stationary phase, the components are separated.
1
Stronger interaction Weaker interaction 2
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PRESENT CHALLENGES
Analysis of matrices like pharmaceutical dosage forms and biological samples will always be challenging, due to their great diversity, intricacy and complexity. Analyzing complex samples like biological products and biological fluids is a significant challenge even with todays advanced and sophisticated instrumentation.
Quality assurance & quality control of pharmaceuticals and formulations play a vital role in ensuring the availability of safe & effective drug products to the population. Quantitative estimation of the chemical entity of a drug substance is pivotal to its quality assurance and control.
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The problem may be a simple one when one is dealing with a pure and single substance. But, during the process of formulation, the original drug substance of high purity is often diluted and mixed with other additives. This may lead to interferences of the additives in the method of estimation. The overall aim of our research is to develop new methods for quantitative determination of novel drugs in pharmaceutical dosage forms. The emphasis is to find new principles for separations using liquid chromatography (HPLC) and to understand the mechanisms
behind.
13
INSTRUMENTATIO N
13
SCHEMATIC REPRESENTATION OF AN HPLC UNIT

1.Solvent reservoirs 2. Solvent degasser 3. Gradient valve 4. Mixing vessel for delivery of the mobile phase 5. Highpressure pump 6.Switching valve in "inject position & Switching valve in "load position 7. Sample injection loop 8.Pre-column(guard column) 9. Analytical column 10. Detector (i.e. IR, UV) 11. Data acquisition 12. Waste or fraction collector
139
BASIC FLOW CHART OF A HPLC SYSTEM SETUP
140
HPLC System Components

Pumps
Micro to Analytical to Preparative Flow Rates Isocratic and Gradient Configurations

Degasser
How it Affects Pumping and Sample Injection

Valves
Solvent Selection and Flow Selection

141
Sample Injection Manual Injector or Autosampler Oven How Temperature Affects Separation Valves for Column Switching Detectors UV-VIS Diode Array Fluorescence Light Scattering Refractive Index Conductivity Mass Spectrometer Recorders and Integrators
142
Fraction Collector Isolate Specific Sample Components Purify Compounds for Multi-Step Synthesis Column Types of Packing Material Factors Affecting Separation Particle Size and Column Length Flow Rate and Temperature
14
A SOLVENT DELIVERY SYSTEM

A mobile phase is pumped under pressure from one or several reservoir and flows through the column at a constant rate.
For NP separation eluting power increases with increasing polarity of the solvent but for reversed phase separation, eluting power decreases with increasing polarity. A degasser is needed to remove dissolved air and other gases from the solvent.
144
HPLC DEGASSING
Degassing removes dissolved air that interferes with check valve operation Refluxing not practicable Ultrasonic degassing ineffective & applicable for ACN/ Water Helium sparge Gas line from the tank directly in the solvent bottle
Vacuum degassing Sonicate before connecting to the system Online with a degassing unit
145
Various solvent delivery systems
14
PUMP MODULES
Types:
Isocratic pump delivers constant mobile phase composition; solvent must be pre-mixed; lowest cost pump Gradient pump delivers variable mobile phase composition; can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase Binary gradient pump delivers two solvents Quaternary gradient pump four solvents
147
The pump is one of the most important component of HPLC, since its performance directly affects retention time, reproducibility and detector sensitivity.
Three main types of pumps are used in HPLC. Displacement pump Reciprocating pump Pneumatic (or) constant pressure pump
148
DISPLACEMENT PUMP: It produce a flow that tends to

independent of viscosity and back pressure and also output is pulse free but possesses limited capacity (250ml).
RECIPROCATING PUMP: It has small internal volume (35-400l), their high output pressure(up to 10,000psi) and their constant flow rates. But it produces a pulsed flow.
PNEUMATIC (OR) CONSTANT PRESSURE PUMP: They are pulse free . Suffer from limited capacity as well as a dependence of flow rate on solvent viscosity and column back pressure. They are limited to pressure less than 2000 psi.
149
HPLC PUMPS TWO BASIC TYPES

Tandem piston
Two pistons with different volumes (48 and 24 L) During each stroke, 24 L of liquid is delivered Best for higher analytical flow rates, up to 10 mL/min Some pulsation is observed, and pulse dampeners are available Not recommended for pulse-sensitive detectors like RID and CDD
150
TANDEM PISTON PUMP

Secondary Piston
Primary Piston
151
DUAL PISTON
Two pistons with equal volume (10 L each) During each stroke, 10 L is delivered Best for low flow rates (< 1 mL/min) Little to NO pulsation
So its ideal for pulse sensitive detectors like RID and CDD
15
DUAL PISTON
153
OTHER PUMP COMPONENTS

Check Valves Control liquid movement in and out of the pump head
154
Piston/plunger seal
Prevents solvent leakage out of pump head
Inline filter
Removes solvent particulates
155
VALVES USED WITH PUMPS

Solvent Selection 2 Solvents Per Pump
Use for solvent switching
. . .
. . .
B 1 2 1 2 1 2
. . .
A A B C
156

Use for pump loading of large sample volumes
Pump B strong gradient solvent. Form the gradient with B.CONC command
Pump A weak gradient solvent and sample loading
12 A B C
Weak gradient solvent
Sample
157

Use for low pressure gradient formation
To Column
Combine any proportion of A/B/C/D.

ABCD
REQUIRES injector.
additional
mixing
before
the
158

Use for different gradients in method development
Pump A
A B B C D A 4 Pairs
Pump B
A B C D B
Pump A
A B C D
Pump B
A, B, C, D A, B, C, D A, B, C, D A, B, C, D
A
16 Combinations
15
SAMPLE INJECTION SYSTEM There are three important ways of introducing

the sample in to the injection port. Loop injection : in which a fixed amount of volume is introduced by making use of fixed volume loop injector. Valve injection: in which, a variable volume is introduced by making use of an injection valve.
On column injection: in which, a variable volume is introduced by means of a syringe through a septum.
160
161
SAMPLE INJECTION MANUAL

Manual Injector with Syringe
Fixed loop of varying sizes (1 to 20 mL or more) Fill with syringes of varying sizes Can include a switch to start a data system
16
SAMPLE INJECTION AUTOMATIC

Fixed-Loop Auto sampler Loop is installed on the valve and can be changed for different injection volumes External syringe draws sample and fills loop
Advantages: low cost rugged few moving parts Disadvantages: Poor performance for low volume injections higher carryover always some sample loss SSJCP, Department of Pharmaceutical Analysis
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Sample Injection how is a sample actually put into an LC system
Manual Injector:
1. User manually loads sample into the injector using a syringe and then turns the handle to inject sample into the flowing mobile phase which transports the sample into the beginning (head) of the column, which is at high pressure
Autosampler:
1. User loads vials filled with sample solution into the autosampler tray (100 samples) and the autosampler automatically : 2. measures the appropriate sample volume, 3. injects the sample, 4. then flushes the injector to be ready for the next sample, etc., until all sample vials are processed for unattended automatic operation
164
SAMPLE INJECTION FIXED LOOP

External syringe draws sample, then fills the fixed-volume loop attached to the valve.
165
Needle-in-the-flow path auto sampler Sample loop and needle are a single piece of tubing Loop and needle are cleaned during the run Metering pump draws sample very precisely Advantages: no sample loss, low carryover
Disadvantages: higher cost more delay volume for gradient
16
SAMPLE INJECTION TO FLOW PATH
Sample Loading
Sample Injection Everything drawn into the needle goes to the column.
16
RINSING AFTER INJECTION
Rinse liquid flows through ports 5 and 6 of the high pressure valve.
Sample aspiration uses port 5.

If air is present around port 5, injection reproducibility will be low. Rinse liquid degassed! MUST be
168
%A {H2O}
Ready
%B %C {MeOH}
Flow Rate Pressure (mL/min) (atmos.)
to column
load inject
Rheodyne Injector
Varian 9010 Solvent Delivery System
to injector
through pump through pulse dampener
Column
Ternary Pump
A B
C
from solvent reservoir
to det ect or
169
CHROMATOGRAPHIC COLUMN
The column is usually made up of heavy glass or stainless steel tubule to withstand high pressure The columns are usually 10-30cm long and 4-10mm inside diameter containing stationary phase at particle diameter of 25m or less Column with internal diameter of 5mm give good results because of compromise between efficiency, sample capacity, and the amount of packaging and solvent required
170
Within the Column is where separation occurs Key Point Proper choice of column is critical for success in HPLC Types of columns in HPLC: Analytical [internal diameter (i.d.) 1.0 - 4.6-mm; lengths 15 250 mm] Preparative (i.d. > 4.6 mm; lengths 50 250 mm) Capillary (i.d. 0.1 - 1.0 mm; various lengths) Nano (i.d. < 0.1 mm, or sometimes stated as < 100 m) Materials of construction for the tubing Stainless steel (the most popular; gives high pressure capabilities) Glass (mostly for biomolecules) PEEK polymer (biocompatible and chemically inert to most SSJCP, Department solvents) of Pharmaceutical Analysis
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HPLC Columns Packing Materials

Columns are packed with small diameter porous particles.
The most popular sizes are: 5-m, 3.5- m and 1.8-m Columns are packed using high-pressure to ensure that they are stable
during use. Most users purchase pre-packed columns to use in their liquid chromatographs
These porous particles in the column usually have a chemically bonded

phase on their surface which interacts with the sample components to separate them from one another for example, C18 is a popular bonded phase
The process of retention of the sample components (often called analytes) is

determined by the choice of column packing and the selection of the mobile phase to push the analytes through the packed column.
172
HPLC COLUMN OVENS

Block heater with solvent preheater Column is housed between 2 metal plates Mobile phase is plumbed into the block for preheating
Forced air Column is in a large chamber with air circulation Better temperature equilibration Room for column switching valves
173
Why Use a Column Oven? Retention times decrease & higher flow rates possible
2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water 50C 45C 40C 35C 30C 25C 20C
174
DETECTORS
The function of detector in HPLC is to monitor the mobile phase as it merges from the column. Detectors are usually of two types: Bulk property detectors: It compares overall changes in a physical property of the mobile phase with and without an eluting solute e.g. refractive index ,dielectric constant or density.
Solute property detectors: It responds to a physical property of the solute which is not exbited by the pure mobile phase.e.g.UV absorbance,fluoroscence or diffusion current. SSJCP, Department of Pharmaceutical Analysis
175
TYPES OF DETECTORS
There are mainly 4 types of detectors are used in HPLC: Photometric detectors. Single wavelength detectors. Multi wavelength detectors. Variable wavelength detectors. Programmable detectors. Diode array detectors . Fluorescence detectors. Refractive index detectors. Electrochemical detectors. Evaporative light scattering detectors IR detectors UV detectors SSJCP, Department of Pharmaceutical Analysis
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PHOTOMETRIC DETECTORS
These normally operate in the ultra violet region of the spectrum .
Most extensively pharmaceutical analysis. used in
177
SINGLE WAVELENGTH DETECTORS
Equipped with a low pressure mercury discharge lamp.

The absorbance is measured at the wavelength of mercury at 254 nm.
178
MULTI WAVELENGTH DETECTORS

Employ mercury and other discharge sources.
When used in combination with interference filters allow a no of monochromatic wavelengths to be selected e.g. 206, 226, 280 , 313, 340 or 365 nm.
179
Multi-wavelength UV-Vis Absorption Detector
Deuterium Lamp
Photodiode Array
18
VARIABLE WAVELENGTH DETECTORS

Use a deuterium light source. A grating monochromator to allow selection of any wavelength in deuterium continuum (190-360 nm).
177
181
UV-VISIBLE DETECTOR
UV-Visible
Wavelength range 190-700 nm D2 and W lamps Most common HPLC detector for a variety of samples Proteins and peptides Organic molecules Pharmaceuticals Monitor two wavelengths at one time
18
UV-Visible Detector
18
Variable wavelength detector
184
Variable UV/Vis Detector

ABS AUFS l RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min
Ready
185
PROGRAMMABLE DETECTORS
Allow the automatic change of wavelength between and during the chromatographic analysis.
186
DIODE ARRAY DETECTORS

They are microprocessor controlled photodiode array spectrophotometers in which light from an UV source passes through the flow cell into a polychromator which disperses the beam so that the full spectrum falls on the array of diodes.
183
187
DIODE ARRAY DETECTOR

Wavelength range 190-900 nm D2 and W lamps
Spectral information about sample Create compound libraries to identify unknowns
Monitor an entire wavelength range at one time up to 790 wavelengths vs. only 2 with a UV detector
188
DIODE ARRAY DETECTOR
189
FLUORESCENCE DETECTOR
These are essentially filter fluorimeter or spectro -fluorimeters equipped with grating monochromators, and micro flow cell. Their sensitivity depends on the fluorescence properties of the components in the elute.
190
Fluorescence detector
Xenon lamp for light source
Excitation wavelength range: 200-650 nm

Emission wavelength range: up to 900 nm depending on photomultiplier installed
Used primarily for amino acid analysis

Derivatize samples before (pre-column) or after separation( post-column)
191
Fluorescence Detector
19
REFRACTIVE INDEX DETECTORS

Which respond to the change in the bulk property of the refractive index of the solution of the component in the mobile solvent system. The sensitivity of the refractive index detector is much less than that of specific solute property detectors, they are useful for the detection of substances(e.g ,carbohydrates & alcohols) which do not exhibit other properties that can be used as the basis for specific detection.
193
Refractive Index Detector

For samples with little or no UV Absorption
Alcohols, sugars, saccharides, fatty acids, polymers Best results when RI of samples is very different from RI of mobile phase
Flow cell is temperature controlled with a double insulated heating block Requires isocratic separations
Requires low pulsation pumps SSJCP, Department of Pharmaceutical Analysis
19
RI BALANCE
Fill sample and reference cell with mobile phase
195
RI ANALYZE
Mobile phase flows through sample side only As the refractive index changes, the image on the photodiode is
deflected or unbalanced, and the difference in current to the photodiode is measured.
19
Refractive Index Detector
19
ELECTROCHEMICAL DETECTORS
These are based on standard electrochemical principles involving amperometry,voltametryand polarography. These detectors are very sensitive for substances that are electroactive ,i.e. those that undergo oxidation or reduction . They have found particular application in the assay of low levels of endogenous catecholamines in biological tissues,pesticides,tryptophan derivatives and many drugs.
198
Electrochemical Detector
19
EVAPORATIVE LIGHT SCATTERING (ELSD)

Also for low or no UV absorbing compounds Sometimes called a Universal detector Requires NO equilibration (unlike RID) Can be used with gradients and volatile buffers (unlike RID) Semi-volatile compounds can be detected at low 20 temperatures
ELSD OPERATION
Column Effluent Nebulizer Nebulization Chamber Nebulizer Gas (Air or Nitrogen)
Analyte
Drift Tube (Heated Zone Evaporation Area)
PMT Light Source Light Scattering Cell Amplifier Signal Output
201
ELSD v/s OTHER DETECTORS

ELSD has higher sensitivity than UV and RID ELSD can be used with gradients, unlike RID
202
CONDUCTIVITY DETECTOR
Flow cell contains 2 electrodes Measure ion amounts in sample REQUIRES low pulsation pumps
Flow cell must be placed in a column oven
20
Use in Environmental and water testing Fl-, Cl- NO3-, PO43-, SO42 Li+, Na+, K+, Mg2+, Cu2+, M-CN complexes Determine organic acids in fruit juice Oxalic, Maleic, Malic, Succinic, Citric
Analyze surfactants Sulfonates, long/short chain ammonium SSJCP, Department of Pharmaceutical Analysis
20
Mass Spectrometer Detector

Separate sample components as ions according to their mass to charge (m/z) ratio Three stages to detection Vaporization: liquid from HPLC column converted to an aerosol Ionization: neutral molecules converted to charged species (either positive or negative) Mass Analysis: filter ions by m/z ratio SSJCP, Department of Pharmaceutical Analysis
20
TWO IONIZIZATION TYPES

APCI: Ionization Atmospheric Pressure Chemical
For molecules up to 1000 Da Singly charges ions Best for analysis of non-polar molecules
ESI: Electrospray Ionization

Can be used for large biopolymers Forms multiply charged ions Best for the analysis of polar molecules, especially pharmaceutical products and proteins
206
MS DETECTOR
Heated capillary Q-array Octapole Electron Multiplier Detector
Orthogonal source geometry
Quadrupole mass analyser
20
FRACTION COLLECTOR Purify raw materials or compounds from synthesis Collect by slope, level, time, volume Isolate single peaks per tube, or divide peaks into small slices for extra purity
20
Temperature Control in HPLC: Why is it needed?

Reproducibility Retention in HPLC is temperature-dependent If temperature varies, then it is difficult to assign peaks to specific compounds in the chromatogram and the peak areas/heights may vary
Solubility Certain chemical compounds may have low solubility in the HPLC mobile phase If they are injected into the flow stream they may precipitate or other difficulties may arise Stability Certain chemical compounds, especially biological compounds such as enzymes or proteins, may not be stable at room temperature or higher The temperature needs to be much lower down to 4C
20
How is Temperature Control Achieved?

Three (3) ways the temperature of a column could be controlled, use: Oven Heater Block Water bath
21
What is HPLC used for?

Separation and analysis of non-volatile or thermally-unstable compounds HPLC is optimum for the separation of chemical and biological compounds that are non-volatile NOTE: If a compound is volatile (i.e. a gas, fragrance, hydrocarbon in gasoline, etc.), gas chromatography is a better separation technique.
211
Typical non-volatile compounds are: Pharmaceuticals like aspirin, ibuprofen, or acetaminophen (Tylenol) Salts like sodium chloride and potassium phosphate Proteins like egg white or blood protein Organic chemicals like polymers (e.g. polystyrene, polyethylene) Heavy hydrocarbons like asphalt or motor oil Many natural products such as ginseng, herbal medicines, plant extracts Thermally unstable compounds such as trinitrotoluene (TNT), enzymes etc.
212
FOR QUALITATIVE ANALYSIS

The identification(ID) of individual compounds in the sample; the most common parameter for compound ID is its retention time (the time it takes for that specific compound to elute from the column after injection); depending on the detector used, compound ID is also based on the chemical structure, molecular weight or some other molecular parameter. 21
FOR QUANTITATIVE ANALYSIS

The measurement of the amount of a compound in a sample
(concentration); meaning, how much is there?
There are two main ways to interpret a chromatogram (i.e.

perform quantification):
determination of the peak height of a chromatographic peak

as measured from the baseline;
determination of the peak area (see figure below); In order to make a quantitative assessment of the
compound, a sample with a known amount of the compound of interest is injected and its peak height or peak area is measured.
In many cases, there is a linear relationship between the

height or area and the amount of sample.
214
Preparation of Pure Compound(s)

By collecting the chromatographic peaks at the exit of the detector and concentrating the compound removing/evaporating the solvent (analyte) by
a pure substance can be prepared for later use (e.g. organic synthesis, clinical studies, toxicology studies, etc.). This methodology chromatography. is called preparative
215
Trace analysis
A trace compound is a compound that is of interest to the analyst but its concentration is very low, usually less than 1% by weight, often parts per million (ppm) or lower; the determination of trace compounds is very important in pharmaceutical, biological, toxicology, and environmental studies since even a trace substance can be harmful or poisonous; in a chromatogram trace substances can be difficult to separate or detect; high resolution separations and very sensitive detectors are required SSJCP, Department of Pharmaceutical Analysis
216
217
SEPARATION TECHNIQUES IN HPLC METHOD DEVELOPMENT
GOAL
Resolution
COMMENT
Precise and rugged quantitative analysis requires that Rs be greater than 1.5 3-10 min is desirable for routine procedures 2% for assays; 5% for less-demanding analyses; 15% for trace analyses Narrow peaks are signal/noise ratios desirable for large
Separation time Quantitation
Peak Height
Solvent composition
Minimum mobile-phase use per run is desirable
218
THE VALIDATION PROCESS

It consists of four distinct steps: Software validation
Hardware (instrumentation) validation/qualification

Method validation System suitability
219
HPLC SYSTEM QUALIFICATION
220
GOALS FOR AN IMPROVED ANALYTICAL METHOD DEVELOPMENT Qualitative identification - structural information, retention time, color change, pH etc Quantitative determination - accurate, precise and reproducible in any laboratory settings Ease of use, viability to be automated, high sample throughput, and rapid sample turnaround time. Decreased cost per analysis - using simple quality assurance and quality control procedures
22
Sample preparation minimizing - time, effort, materials, and volume of sample consumed Direct output of qualitative or quantitative data evaluations, interpretation, printing out and transmission
OPTIMIZATION & ANALYTICAL FIGURES OF MERIT

initial sets of conditions - resolution, peak shape, plate counts, asymmetry, capacity, elution time, detection limits quantifying the specific analyte of interest, accuracy and precision of Quantitation and specificity must be defined. SSJCP, Department of Pharmaceutical Analysis
222
Chromatographic resolution adequate Limits of detections are lower Calibration plots are linear Sample throughout is increased
Sample preparation before analysis is minimized

Interference is minimized and identified Data acquisition - translated, interpreted, printed & stored Reproducibility of analytical figures of merit & Cost per analysis is minimized
223
METHOD VALIDATION APPROACHES Samples of the given analyte

Concentration in the matrix
High degree of accuracy and precision Zero, Single and Double Blind spiking methods Inter laboratory collaborative studies Comparison with a currently accepted compendium method
224
STEP-BY-STEP HPLC METHOD DEVELOPMENT, OPTIMIZATION AND VALIDATION: AN OUTLINE Analyte Standard Characterization
Method Requirements Literature Search and Prior Methodology
Choosing a Method
Instrument Setup and Initial Studies Optimization Demonstration of Analytical Figures of Merit with Standards SSJCP, Department of Pharmaceutical Analysis
225
Evaluation of Method Development with Actual Samples and Derivation of Figures of Merit Validation of Figures of Merit Determination of Percent Recovery of Actual Sample and Demonstration of Quantitative Sample Analysis Method Validation Preparation of Written Protocols and Procedures Transfer of Method Technology to Outside Laboratories and Interlaboratory Collaborative Studies
22
Comparison of Interlaboratory Collaborative Studies Preparation of Summary Report on Overall Method Validation Results Summary Report of Final Method and Validation Procedures and Results and also Preparation of Journal Article for Submission
THE OUTLINE PROTOCOL OF HPLC METHOD
22
STEPS FOR HPLC METHOD DEVELOPMENT

Information on sample, define separation goals Validate method for release to routine laboratory
Need for special procedure sample pretreatment, etc
Quantitative calibration
Choose detector and detector settings
Check for problems or requirement for special procedure
Choose LC method; preliminary run; estimate the best separation conditions
Optimize separation conditions
228
PARAMETERS USED IN METHOD VALIDATION
229
SPECIFICITY
It is the ability to measure accurately and specifically the analyte of interest in the presence of other components that may be expected to be present in the sample matrix Specificity is also measured and documented in a separation by the resolution, plate count (efficiency) and tailing factor Blank solution to show no interference with excipients or degradation products or impurities
Placebo to demonstrate the lack of interference from excipients Spiked samples to show that all known related substances are resolved from each other SSJCP, Department of Pharmaceutical Analysis
23
LINEARITY AND RANGE

It is the ability of the method to elicit test results that are directly proportional to analyte concentration within a given range Reported as the variance of the slope of the regression line ICH guidelines specify a minimum of five concentration levels Assay : 80-120% of the theoretical content of active Content Uniformity: 70-130% Dissolution: 20% of limits; e.g if limits cover from 20% to 90% l.c. (controlled release), linearity should cover 0-110% of l.c. SSJCP, Department of Pharmaceutical Analysis
231
Impurities: reporting level to 120% of shelf life limit

Assay/Purity by a single method: reporting level of the impurities to 120% of assay limit Correlation coefficient (r) = API: 0.998 & Impurities: 0.99 y-intercept and slope should be indicated together with plot of the data
232
ACCURACY
Measure of exactness of an analytical method or closeness of agreement between the measured value and the value that is accepted either as a conventional, true value or an accepted reference value
Measured as percentage of analyte recovered by assay, by spiking samples in a blind study API (Active Pharmaceutical Ingredient): against an RS (Reference Standard) of known purity, or via an alternate method of known accuracy; analysis in triplicate FPP (Finished Pharmaceutical Product): samples/placeboes spiked with API, across the range of 80-120% of the target concentration, 3 concentrations, in triplicate each SSJCP, Department of Pharmaceutical Analysis
23
Report % recovery (mean result and RSD): 1002% Impurities: API/FPP spiked with known impurities
Across the range of LOQ-150% of the target concentration (shelf life limit), 3-5 concentrations, in triplicate each. (LOQ, 50%, 100%, 150%)
% recovery: in general, within 80-120%, depends on the level of limit
ICH Q2 states: accuracy may be inferred once precision,
linearity and specificity

234
LOD / LOQ
LOD: the lowest concentration of an analyte in a sample that can be detected though not necessarily quantitated.
LOQ: the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy under the stated operational conditions of the method
signal to noise ratio: LOD = 3:1 , LOQ = 10:1

May vary with lamp aging, model/manufacturer of detector, column
standard deviation of the response and the slope of the calibration curve at levels approximating the LOD /LOQ
= the standard deviation of the response, based on the standard deviation of the blank & the calibration curve & S = Slope
235
should be validated by analysis of samples at the limits LOD: below the reporting threshold
LOQ: at or below the specified limit

Not required for assay/dissolution methods Applicant should provide the method of determination the limits chromotograms
23
ROBUSTNESS / RUGGEDNESS Robustness: capacity of a method to remain unaffected by small deliberate variations in the method parameters Ruggedness: degree of reproducibility of the results obtained under a variety of conditions, expressed as % RSD Evaluated by varying method parameters such as percent organic solvent, pH, ionic strength or temperature , determining the effect on the results of the method, columns, laboratories, analysts, instruments, reagents and experimental periods. 23
SYSTEM SUITABILITY TESTING (SST)

used to verify resolution, column efficiency, and repeatability of the analysis system to ensure its adequacy for performing the intended application on a daily basis.
Parameters: Number of theoretical plates (efficiency) Capacity factor Separation (relative retention) Resolution Tailing factor Relative Standard Deviation (Precision)
238
Center for Drug Evaluation and Research (CDER) Limits for SST
239
CHARACTERISTICS TO BE VALIDATED IN HPLC CHARACTERISTICS ACCEPTANCE CRITERIA

Accuracy/trueness Repeatability Recovery 98-102% with 80, 100 & 120% spiked sample RSD < 2%
Intermediate precision
Specificity/selectivity Detection limit Quantitation limit Linearity Range Stability of sample solution
RSD < 2%
No interference S/N > 2 or 3 S/N > 10 Correlation coefficient r > 0.999 80 120% > 24 hours or > 12 hours
24
TYPICAL HPLC INSTRUMENT TEST ITEM VERIFICATION USER LIMIT REPORT ACTUAL LIMIT
DAD noise Baseline drift DAD WL calibration DAD linearity Pump performance Temperature (column heater) stability < 5 X 10-5 AU < 2x 10-3 AU/hour 1 nm 1.5 AU < 0.3% RSD RT 1 X 10-5 AU 1.5 X 10-4 AU/hour 1 nm 2.2 AU 0.15% RSD RT 0.15 C
0.15 C
Precision of peak area
0.5% RSD
0.09% RSD
241
METHOD VALIDATION PROTOCOL

1. On day 1, a linearity test over 5 levels for both the drug substance (bulk) and dosage form is performed 2. Comparison of the results between the drug substance and dosage form fulfills the accuracy requirement 3. At the end of day 1, 6 repetitions are performed at 100% of the drug substance for repeatability 4. Steps 1 and 2 are repeated over 2 additional days for intermediate precision 5. LOQ is evaluated by analyzing the drug substance over 5 levels, plus 6 repetitions for precision 6. Baseline noise is evaluated over 6 repetitions of blank injections for the determination of LOD.
242
TROUBLE SHOOTING
ASK PULLA
(TIPS & FACTS)
For any further clarification or details of the below content(s) feel free to mail me :
24
1.What is HPLC anyway? 2. How to become friendly with your HPLC equipment? 3. How to get started? 4. Which column do I have to install in the HPLC instrument? 5. How do I prepare a mobile phase? 6. What is the requirement of equilibrating the system before the advent of sample preparation. 7. What do I have to pay attention to before starting a measurement? 8. How do I start working with the HPLC equipment? 9. What's the reason for quitting your HPLC system?
244
SIMPLE TESTS & DECISION CRITERIA

10.What does the name of a column tell us? 11. Is this C18 column the right choice for my sample? 12. Why are polar solutes well separated with one C18 column and hardly at all with another? 13. How can I clean the RP Phase quickly? 14. How best do I degas my mobile phase? 15. Methanol or Acetonitrile? Best choice of solvent..? 16. The pH of the mobile phase too high or too low. What can I do? 17.What is the right ionic strength of the buffer? 18.How to make sense of the dead volume of an isocratic apparatus? 19.Producing a gradient chromatogram influence of instrumentation? SSJCP, Department of Pharmaceutical Analysis 20. Does the pump work correctly, precisely or accurately?
245
21. How to test an HPLC instrument and its modules? 22. Injections of solutes as an aqueous solutions? 23. What is the largest tolerable injection volume? 24 . How critical are the temperature changes? 25. How to choose HPLC equipment and a supplier? 26. Is the current method a robust one?
PROBLEMS & THEIR SOLUTIONS

27. Sample preparation how critical are which mistakes? 28. Flushing of an HPLC equipment? 29. Dirt in the UV detection cell? 30. The lamp is new what happened to the peak? 31. What are the causes of pressure changes or deviations? SSJCP, Department of Pharmaceutical Analysis
246
32. Is the right or the left pump head defective? 33. Baseline noise and damping? 34. The retention times increase- is it the pump or the M.P ? 35. Which buffer is right for which pH? 36. An interesting alternative for the separation of acids & bases with a buffer.. 37. What can be the reasons for a change in retention times? 38. I use up a lot of RP columns; what should I do? 39. Why does my NP system not work any more? 40. Chemical tailing at the presence of metal ions? 41. How to avoid memory effects? 42. How do the default values on my PC affect the resolution?
247
TIPS TO OPTIMIZE THE SEPARATION

43. Which is the right injection techniques to get sharper peaks? 44. My peaks appear too early how can I move them in an RP system to later retention times? 45. How can I increase the plate number? 46. Limit of detection: How can I see more? 47. How can I speed up a separation? 48. How can I optimize a separation? 49. Dead volume capacity, capacity factor, selectivity how can I use them in everyday life? 50. Which flow is optimal for me? 51. How can I optimize a gradient elution? 52. Separation of ionic solutes? What works out best end capped phases, inert phases, phosphate buffer or ion pairing reagents? SSJCP, Department of Pharmaceutical Analysis
248
SITUATION/SYMPTOM/CAUSE
EQULIBRATION 53. SLOW COLUMN
RP- Ion pairing long chain
54.VARYING / VARIABLE TIMES
RETENTION
gradient insufficient column regeneration time ion pairing insufficient equilibration time isocratic insufficient equilibration time irregular column equilibration time Leak change in M.P composition air trapped in pump SSJCP, Department of Pharmaceutical Analysis
249
buffer capacity insufficient contamination buildup equilibration time insufficient for gradient run or changes in isocratic M.P first few injections active sites inconsistent online M.P mixing or delivery selective evaporation of M.P component varying column temperature check valve malfunctioning pump cavitations, phase collapse (de-wetting process) Column temperature fluctuations First few injections adsorption on active sites column overloading sample solvent incompatible with M.P SSJCP, Department of Pharmaceutical Analysis
250
Column problem improper M.P column aging
55. INCREASED RETENTION TIME

decreasing flow rate, changing M.P composition, loss of bonded S.P, active sites on column packing Low M.P flow rate Column temperature low Improper gradient setting Column activity increasing System not equilibrated SSJCP, Department of Pharmaceutical Analysis
25
M.P removing water from LSC column Incorrect M.P Loss of bonded S.P M.P composition changing Active sites on silica packing
56. DECREASED RETENTION TIME

column overloaded with sample increasing flow rates loss of bonded S.P or base silica from column column aging, basic compounds pH too low High M.P flow rate Column temperature high SSJCP, Department of Pharmaceutical Analysis
25
Improper gradient Incorrect M.P Column activity decreased System not equilibrated Deactivation by strongly retained garbage Too strong sample solvent
57. RETENTION BEYOND TOTAL PERMEATION VOLUMES

SEC solute interaction with S.P.
25
58. LOSS OF RESOLUTION

M.P contaminated/deteriorated Obstructed guard or analytical column Column overload with sample Degraded column Column not fully equilibrated Loss of S.P from the column Dirty column Loss of column liquid phase Distorted column bed Wrong column or M.P
254
SENSITIVITY 59. Lack of sensitivity

auto sampler flow lines blocked detector attenuation set too high first few samples injections sample adsorption in injector sample loop or column injector sample loop under filled not enough sample injected peak signals are outside detectors linear range sample losses during sample preparation sample losses on column peak too broad
255
BASELINE
60. Distribution At Void
air bubbles in M.P positive-negative differences in RI of injection solvent & M.P
61. BASELINE DRIFT

Column temperature fluctuations Non homogeneous M.P Contaminant or air buildup in detector, sample or reference cell Plugged outlet line after detector M.P mixing problem or change in flow rate Slow column equilibration when changing M.P M.P contaminated or deteriorated or not prepared from high quality chemicals SSJCP, Department of Pharmaceutical Analysis
25
Strongly retained materials in sample can elute as very broad peaks and appear to be a rising baseline Detector not set at absorbance maximum but at slope of curve M.P or sample vaporizing Failing detector source Detector problem Solvent immiscibility Contamination bleed in system Solvent demixing Slow change in pump output Partial plugging of injection port or sample valve or column inlet by particulate matter Contaminated or bleed column Contamination in detector cell SSJCP, Department of Pharmaceutical Analysis
25
Change in detector temperature Malfunction of detector source Contamination in solvent reservoir Previous M.P not removed Negative direction Positive direction
62. BASELINE NOISE (REGULAR)

Air in M.P or detector cell or pump Pump pulsations Incomplete M.P mixing Temperature effect Other electronic equipment on same line Leak or partial blockage of loop injector valve or detector lamp problem Dirty flow cell
258
63. BASELINE NOISE (IRREGULAR)

Leak M.P contaminated or deteriorated or prepared from low quality materials Detector or recorder electronics Air trapped in system Air bubbles in detector Detector cell contaminated Weak detector lamp Column leaking silica or packing material or column packing passing through detector Continuous detector lamp problem or dirty in the flow cell gradient or isocratic proportioning - lack of solvent mixing & malfunctioning proportioning valves SSJCP, Department of Pharmaceutical Analysis
259
occasional sharp spikes, external electric interferences, periodic pump pulse, random contamination buildup, spikes bubble in detector & column temperature higher than B.P of solvent
RECOVERY
64. POOR SAMPLE RECOVERY
absorption or adsorption of proteins adsorption on column packing absorption on tubing and other hardware components chemisorption on column packing hydrophobic interactions between S.P & biomolecules SSJCP, Department of Pharmaceutical Analysis
260
less than 90% yield for acidic compounds irreversible adsorption on active sites less than 90% yield for basic compounds irreversible adsorption on active sites
LEAKS
65. LEAKY FITTING
A loose fitting Stripped fitting Over tighten fitting Dirty fitting Mismatched part/fitting SSJCP, Department of Pharmaceutical Analysis
261
66. LEAKS AT PUMP

Loose check valve Mixer seal failure Pump seal failure Pressure transducer failure Pulse damper failure Proportioning valve failure
67. INJECTOR LEAKS

Rotor seal failure Blocked loop Loose injection port seal Improper syringe needle diameter Waste line siphoning Waste line blockage SSJCP, Department of Pharmaceutical Analysis
26
68. COLUMN
LEAKS
Loose end fittings Column packing in ferrule Improper frit thickness 69. DETECTOR
LEAKS
Cell gasket failure Cracked cell window Leaky fittings Blocked waste line
263
PROBLEMS DETECTED BY SMELL, SIGHT & SOUND

70. SOLVENT SMELL Leak Spill 71. HOT SMELL Overheating 72. ABNORMAL METER READING Pressure abnormality Column oven Detector lamp failing 73. WARNING LAMP Pressure limits exceeded Other warning signals SSJCP, Department of Pharmaceutical Analysis
26
RETENTION BEYOND TOTAL PERMEATION VOLUMES

74. SEC solute interaction with S.P.
75. NO FLOW
Pump off flow interrupted/obstructed/ blocked leak air trapped in pump head insufficient mobile phase faulty check valves
PRESSURE
76. DECREASING PRESSURE insufficient flow from pump leak in hydraulic lines from pump to column SSJCP, Department ofor Pharmaceutical Analysis leaking pump check valve seals, pump cavitations
265
77. FLUCTUATING PRESSURE

bubble in pump leaking pump check valve or seals
78. HIGH BACK PRESSURE

column blocked with irreversibly adsorbed sample column particle size too small microbial growth on column M.P viscosity too high plugged frit in line filter or guard column plugged column inlet frit polymeric columns- solvents change causes swelling of packing salt precipitation blockage in injector when disconnected from column SSJCP, Department of Pharmaceutical Analysis
26
79. INCREASING PRESSURE blocked flow lines particulate buildup at head of column water organic solvent systems buffer precipitation
80. NO PRESSURE/PRESSURE LOWER THAN USUAL OR ERRATIC/FLUCTUATING COLUMN PRESSURE

Leak in high pressure system leakage in flow line M.P flow interrupted/obstructed, air trapped in pump head leak at column inlet end fitting, air trapped elsewhere in system worm pump seal causing leaks around pump head dirt in pump check valve SSJCP, Department of Pharmaceutical Analysis
26
pump starvation inlet tube connection is leaking pump head has deteriorated insufficient degassing of solvent plunger movement is abnormal sound on metal on metal can be heard drain valve is open insufficient flow from/to column flow rate too low column temperature too high improper column
26
81. PRESSURE HIGHER THAN USUAL

Problem in pump or injector or tubing
partial obstructed pre- column or guard column or analytical column partial blockage in detector cell or detector inlet downstream side of pump is clogged line filter is clogged column particle size too small M.P viscosity too high microbial growth in column salt precipitation in RP internal damper is clogged or internal line is clogged inside diameter of tubing is excessively small
improper M.P or column

column temperature too low flow rate too high
26
82. SOLVENT DELIVERY IS UNSTABLE/FLUCTUATION IN PRESSURE IS LARGE

Bubbles in pump chamber old solvent remaining in pump chamber bubbles in the suction filter enter the pump malfunctioning of check valve leak in the flow line flow line is clogged
83. PUMP OPERATING BUT NO SOLVENT IS DELIVERED

Bubbles in the pump chamber Air enters from the joints between suction filter or inlet pipe. SSJCP, Department of Pharmaceutical Analysis
27
84. MEASURED FLOW RATE IS LOWER THAN THE SET FLOW RATE
Malfunctioning of check valve clogged suction filter
PEAKS
85. NO PEAKS / VERY SMALL PEAKS instrumental problem or wrong M.P or S.P Detector lamp off loose /broken wire between detector and integrator or recorder no M.P flow no sample or deteriorated sample or wrong sample Setting too high on detector or recorder SSJCP, Department of Pharmaceutical Analysis
27
86. SPLIT PEAKS/DISTORTED PEAKS/DOUBLE PEAKS

Contamination on guard or analytical column inlet Partially blocked frit Small void at column inlet Sample solvent incompatible with M.P Inhomogeneous bed structure or channeling Injection solvent too strong Sample volume to large
87. PEAKS INJECTIONS
TAIL IN INITIAL
AND LATER
Sample reacting with active sites Wrong M.P pH or column type or injection solvent Small void at column inlet SSJCP, Department of Pharmaceutical Analysis
27
88. PEAK TAILING

basic solutes silanol interactions beginning of peak doubling chelating solutes - trace metals in base silica silica based column degradation at high pH degradation at high temperature unswept dead volume void formation at head of column interfering co elution peak Guard or analytical column contaminated or worn out M.P contaminated or deteriorated Interference in sample trace metals in base silica
273
89. FRONTING PEAKS

Column overload Sample solvent incompatible with M.P Shoulder or granular baseline rise before a main peak my be another sample component Channeling in column
90. ROUNDED PEAKS

Detector operating outside linear dynamic range Recorder gain too slow Column overloaded Sample column interaction Detector or recorder time constants are set too high Column dried out at ends Contamination on detector cell window SSJCP, Department of Pharmaceutical Analysis
27
91. NEGATIVE PEAKS

RI detection RI of solutes less than of M.P UV absorbance detection absorbance of solute less than that of M.P at a wavelength of choice occurs at void volume Recorder leads reversed sample solvent & M.P differ greatly in composition
92. GHOST/SPURIOUS/VACANT PEAKS

contamination, elution of analytes retained from previous injections Ion Pair Chromatography upset equilibrium oxidation of trifluoroacetic acid in peptide mapping RPC contamination of water unknown interferences in sample Contamination in injector or column SSJCP, Department of Pharmaceutical Analysis
275
Late eluting peak present in sample Air dissolved in sample Elution of sample solvent Impurities in solvent used during gradient Spike bubbles in M.P IPC upsets equilibrium Dirty M.P
93. SPIKES
Bubbles in M.P Column stored without caps Electric interference
27
94. BROAD PEAKS

analyte eluted early due to sample overloaded detector cell volume too large injection volume too large, large extra column volume M.P solvent viscosity too high peak dispersion in injector valve poor column efficiency retention time too long sampling rate of data system too low slow detection time constant, some peaks broad late elution of analytes retained from previous injection high molecular weight compounds on small pore column M.P composition changed M.P flow rate too low
277
Leak between column and detector Extra column effects column overloaded, detector response time or cell volume too large Buffer concentration too low, recorder response time too high, tubing between column & detector too long or ID too large Guard column /column contaminated or worn out Low plate number Void at column inlet Peak represents two or more poorly resolved compounds Column temperature too low Injection volume too large Large extra column volume Long RT
27
95. PEAK DOUBLING

blocked frit co elution of interfering compound co elution of interfering compound from previous injection column overloaded column void or channeling injection solvent too strong sample volume too large unswept injector flow path sample solvent incompatible with M.P.
279
96. CHANGE IN PEAK HEIGHT ( ONE OR MORE PEAKS)

One or more sample components deteriorated or column activity changed Leak especially between injection port and column inlet Inconsistent sample volume Detector or recorder setting changed Weak detector lamp Contamination in detector cell
280
97. CHANGE IN SELECTIVITY

Increase or decrease solvent ionic strength, pH or additive concentration Column changed- new column has different selectivity from old column Sample injected in incorrect solvent or excessive amount of strong solvent Column temperature change
98. LOW SENSITIVITY

Inadequate flow rate Sample not compatible with detector Insufficient sample Sample not eluting from column SSJCP, Department of Pharmaceutical Analysis
281
Dirty detector cell windows Gas bubbles in detector cell Detector attenuation too high Detector or recorder out of calibration Failing or faulty detector source Recorder in wrong millivolt range Sample losses during sample preparation Peaks are outside detectors linearity range Injector sample loop under filled Auto sample flow lines blocked Sample losses in column
99.RECORDER WILL NOT ZERO

Bubbles in UV reference cell or wrong solvent in RI reference cell M.P has not equilibrated with column SSJCP, Department of Pharmaceutical Analysis
28
M.P has not equilibrated with detector or is not compatible with detector Column bleed Contamination bleed from column Previous M.P still in the system Detector not connected to recorder Detector source lamp failing or faulty Dirty detector cell windows Particulates in detector cell Electronic problem with detector Recorder or detector not plugged in or turned on Recorder improperly zeroed Recorder calibration knob out of position
283
100. PROBLEMS RELATED TO ASSAY TYPE EFFECT SITUATION/SYMPTOM CAUSE ANALYSIS

1. Irreproducible sample volume 2. Improper detector response 1. Poor peak reproducibility height or recorder
With all peaks
3. Column or detector is sample overloaded 4. Calibration & sample solutions decomposing 5. Internal standard decomposing or reacting 6. Irreproducible RT
As all above (read cause 1)

2. With symmetrical peaks With all peaks Sample decomposition in column
284
EFFECT
SITUATION/SYMPTOM
CAUSE
As above (read cause 1) Undesired interaction of solute with S.P Poorly packed column Sample column decomposition in
3. With unsymmetrical peaks With all peaks
As above (read cause 1 ) With all peaks 4. Poor peak reproducibility area Improper integrator computer response Internal decomposing With symmetrical peaks or
standard
As above (read causes 1 & 2) As above (read causes 1 & 3)
With unsymmetrical peaks
285
EFFECT
SITUATION/SYMPTOM
CAUSE
Improperly made standards
Calibration or sample solution decomposing Unknown interference with Analytical results higher than peaks for desired components theoretical Internal standard reacting or decomposing Component of interest internal standard too close or
5. Poor analytical accuracy
Improperly made standards

Analytical results lower than Calibration and or solution decomposing theoretical Unknown interference internal standard peak sample with
286
EFFECT
SITUATION/SYMPTOM
CAUSE
Desired decomposing component
No peak seen for very small sample 6. Peak height or peak area calibrations do not intercept zero Improper peak measurement Strange peak seen at or near Peak overlap R.T of component of interest Peak seen in control or blank Contaminated injector run Non linear detector response 7. Variable analytical Non linear calibration plot or accuracy as function of calibration factor Sample component or concentration internal standard decomposing size
287
EFFECT
SITUATION/SYMPTOM
CAUSE
Less precise method used calibration
Analytical variability too 8. Analytical precision large even though proper lower precision with internal insufficient for need standard procedure followed Equipment deficiencies Gradient elution used Low column plate count 9. Analytical insufficient for needs speed Higher analysis speed required for routine analysis Low column selectivity or process analysis Resolution of components too good
28
EFFECT
SITUATION/SYMPTOM
CAUSE
Poor reproducibility separation
Column irreproducibility
10. Difficult to determine Irreproducible sample profile, which peak is to be measured peak sequences or R.T Ghost peaks
Spurious peaks in calibration solutions due to sample decomposition
Peak retention affected by large concentration of unknown 11. Variable analytical results Changing calibration factor S As above (read cause 2)
289
101. PROBLEMS RELATED TO TRACE ANALYSIS

EFFECT SITUATION/SYMPTO M CAUSE
Insufficient detector response Improper detector Sample volume too small Poor detectability for peaks 11. Insufficient sensitivity for of interest needs K too large Column too long Value for too small N too small Separation method not optimized Poor detection when sample volume is limiting Column I.D is too large
29
EFFECT
SITUATION/SYMPTO M
CAUSE
Use of peak area method
Overlap of trace peak with Insufficient resolution interferences 12. Poor trace analysis accuracy or precision Interference from spurious unknown peaks Poor recoveries with sample Variable loss of components pretreatment of interest Change in calibration factor As above (read cause 1)
13. Analysis time too long
Higher analysis speed required for routine analysis As above (read cause 9) or process analysis
291
EFFECT
SITUATION/SYMPTO M
CAUSE
Detector drift
Solvent contamination Base line drift measurement of difficult makes Broad peaks from previous peaks runs are eluting during the analysis Flow rate change
Gradient used at detector sensitivity

Column bleed As above (read situation 9) 14. Analysis time too long Components eluting after peak of interest long
higher
As above (read cause 9) Improper separation system Simple isocratic system used Too complex sample
292
102. Retention of ionizable components in RP-HPLC have to analyzed and studied before start of work 103. Analyte ionization & HPLC retention ? 104. Factors that should be considered prior to method development 105. Drawbacks that may be encountered when working at high pH 106. If it is necessary to work at high pH because compounds are known to degrade at low pH, what could be done to obtain a more rugged method? 107. I know that I do not have any ionic or ionizable components in my mixture..what next? 108. My mixture contain weak acids and weak bases? 109. What may happen if I analyze my weak basic or acidic components at low pH (OR) at high pH? 110. Are there any other drawbacks if I analyze my weak acidic components at high pH? SSJCP, Department of Pharmaceutical Analysis
293
111. My components are weak acids and decreasing pH improves the peak shape but significantly increases the retention, up to one hour for some analysis. How do I decrease the R.T? 112. I do not know the components of this mixture, but I have to develop a method as soon as possible. How do I start my method development? 113. How can the conditions be optimized in order to obtain the desired resolution between components? 114. What is the cause of the retention of the basic compounds upon lowering the pH? 115. Do other acidic modifiers used to adjust the pH of the mobile phase affect the retention of the protonated basic analytes? 116. What are the properties of the acid that may affect the solvation of the basic analytes? 117. Solvation & ionization of the acids- H3PO4, CF3COOH & HClO4? SSJCP, Department of Pharmaceutical Analysis
294
118. What are chaotropic counter anions? 119. How does the use of different modifiers affect the retention of a basic analyte? 120. Is the increase in retention for a protonated basic compound due to a decrease in pH or an increase in acidic modifier counter anion concentration? 121. How different is the chaotropic effect for the different analytes? 122. At a certain pH when different acidic modifiers are employed the counter anion concentrations are not the same. How can one compare the effect of the chaotropic counter anion on the retention of basic analytes? 123. How is the concentration of the a counter anion of a strong acid or weak acid calculated? 124. How do you calculate the concentration of a polyprotic acid such as phosphoric acid? SSJCP, Department of Pharmaceutical Analysis
29
125. If we have certain concentration of phosphate buffer and then adjust with phosphoric acid how do we calculate the amount of H2PO4- present? 126. Is there a way to maintain the pH and simply adjust the concentration of the counter anion of the acidic modifier? 127. The sodium perchlorate salt was added, but what other salts that contain chaotropic counter anion could I use? 128. The chaotropic effect is predominant under highly aqueous conditions, but is it significant where the organic content is higher? 129. What is the influence of temperature on the chaotropic effect? 130. What is the optimal temperature at which method development should be carried out? 131. Is there a way to calculate the pka of my compound within a mixture if only a limited amount is available? 132. How significant will the retention shift be if a change of the counter anion concentration occurs in a low pH region? SSJCP, Department of Pharmaceutical Analysis
290
COMMON IMPURITIES IN HPLC SOLVENTS

CONTAMINATION SOURCE SYMPTOMS Blockage of in line filters, accumulation of dirt over column leading to change in k value, decrease in selectivity, spurious peaks, drifting of peaks, irreversible adsorption of contaminants over column packing resulting in shortening of column life
Particulate matter
Unclean vessel
Water alcohols Peroxides
Suring solvent preparation or improperly dried Stability of bonded phases glassware's Behave like water Degradation Reaction with column packing sample,
297
Dissolved oxygen BHT
Solvent preparation Antioxidant in THF
Degradation UV absorbing
Bacterial or fungal Prolonged storage growth
Can block the in line filters, column frit and degradation of bonded phases
Halogens
Chlorinated solvents Attack stainless steel can form HBr & or part of the system HCl
298
ANALYST QUOTES
No one is ever born talented, nor is talent lucky coincidence, it is acquired through ones own hard endeavor. If I give you a penny, you will be one penny richer and I will be one penny poorer, but if I give you an idea, you will have a new idea, but I shall still have it, too. Compliment your existing knowledge and enhance your skill to ensure that your laboratory can meet to the challenge of today and tomorrow. Established and validated analytical methods should not be changed if there are no reasons that make this absolutely necessary e.g. economic and ecological, even if such a change is possible without any difficulty. Learning is input, performance is output. Learning theory, leads to experimental designs. Plagiarism is stealing from one person and research is borrowing from many. Repetition is a powerful tool, not a sign of incipient senility. Pass on the experience and knowledge to those wanting to learn. Think logically about the system and chemistry involved, you will be surprised how well you are able to predict and control the separation. Be determined, no analytical problem will ever defy solution. There is no barrier to the acquisition of the knowledge. Nobody owns it, everybody partakes of it, and the world becomes richer. Imagination is more important than knowledge.
299
Objective of any test procedure should be to demonstrate that it is adequate for its intended use. Ideas have endurance without death. Have vision, not sight; those who just see and do not strive, bury their talent. Important in reality is not he who had the idea, but he who first expressed it better. Read, write and digest. No book can be a substitute for the inspiration acquired by actual experimentation. No method of analysis is so precise, that provides analysts with recovery values free of error. No drug can be put into dosage form without some compounding error. One good instrument may do the work of fifty ordinary analysts; no instrument can do the work of one extra-ordinary analyst. Art of analysis is mastered in the laboratory, experience is the best teacher. I hear to forget, I see to remember, I experiment to understand. Do not think, expensive equipments will make up for lack of talent, altitude or practical skill. Success is not about being intelligent; it is all about what you do with the intelligence. There is a real connection between hand and the brain, so you must write things as you go along. Knowledge and talent do not age. Perfection is a matter of trial and error, arduous the trial, innumerate the errors.
300
An ordinary teacher tells, good teacher explains, superior teacher demonstrates and the great teacher inspires. Very few people really appreciate others work. There are very rare people who enjoy others success. No method of analysis is so precise to be free of error. What technology may not solve, the idea may. Even the highly trained analysts may have little mastery over the fundamental laboratory procedures. Whatever your unique talents are, you must resolve to discover, use and share them. That which you share will multiply and that which withhold will diminish. A man would do nothing, if he waited until he could do it so well, that is no one would find fault, with what he has done. Dont underestimate the value of experience. All is not done In every age some people have felt, that there is little left to be done, all the really stuff is behind us and all we can hope for is to mop up some details; we wont be able to break new grounds, but every age has been dead wrong in this notation. By contrast, the galloping pace of scientific discovery continues and we learn more every day, every year and not just the details. Nothing dies faster than a new idea is closed mind; let us evolve idea into reality. Have inquiring mind, try to understand and evaluate unexpected laboratory results scientifically and critically. Dont fall in love with your own work or skill to the exclusion f other good ideas.
301
Thanks not those faithful who simply praise words and actions but those who kindly reprove the fault. One cant do inspired analysis without aesthetic qualities. I have never used my knowledge as a vehicle but only as means of sharing my experience with you. Be determined that the method can and shall be devised; no analytical problem will ever defy solution. An analyst has to be in love with analysis without which it is virtually impossible to pass on the knowledge and experience one has to those wanting to learn. Essence of analysis--- weighing--- measuring--- calculations---interpretation If from any art, you take away which concerns weighing, measuring and arithmetic, how little is left of that art. Success story of analysis--- validated analytical instruments--- validated analysts--- validated analytical methods One of the most frustrating aspects for an analyst is working with an ill-defined, poorly designed and invalidated analytical method. It is rather impossible to familiarize oneself with all the possibilities by ones own experience or study, one is usually tempted to apply the methods, those are well established. One of the most difficult situations that an analyst may be confronted with is the selection of suitable analytical method for a particular problem.
302
Would you trust the analytical results from your laboratory if your life depends on them- your answer should be emphatically YES. Analysts are not merely the measuring tape wielders but equal partners, often troublesome as their results cost money. Let the analysts be on guard against any analytical non-sense. It is better to do a modest amount of validation work on as many cases as possible rather than do an exhaustive job on few and no work on other. A good planning is half the job done.
303
304

Ravi Pratap Pulla: Asso - Professor, SSJ College of Pharmacy, V.N.Pally, Gandipet, Hyderabad-75

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Ravi Pratap Pulla: Asso - Professor, SSJ College of Pharmacy, V.N.Pally, Gandipet, Hyderabad-75

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HPLC

RAVI PRATAP PULLA

Asso.Professor, SSJ College of Pharmacy, V.N.Pally, Gandipet, Hyderabad-75.

HPLC THE DEVELOPMENT OF A NAME

SSJCP, Department of Pharmaceutical Analysis

Introduction to Liquid Chromatography

SSJCP, Department of Pharmaceutical Analysis

A Brief History of Chromatography

CHROMATOGRAPHY IS INCOMPLETE WITHOUT LEARNING FEW BASIC TERMINOLOGIES

Column plate number

SSJCP, Department of Pharmaceutical Analysis

Hydrophobic interaction chromatography (HIC) Ion chromatography

Ion exclusion Ion moderated partioning chromatography (IMPC)

Ion pair chromatography (IPC)

Ion exchange chromatography

SSJCP, Department of Pharmaceutical Analysis

Peak width Phase ratio

Retention factor (k) Retention volume (VR or tR)

Plate height (H)

Separation factor (a)

Reduced mobile phase velocity (n) Stationary phase Tailing

Reduced plate height (h)

Interparticle time (tZ)

Capacity factor (k)

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Adsorption chromatography Back pressure

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Detector sensitivity Electrochemical detector

Differential Refractive Index( RI) Elution order

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Organic modifier Partially resolved peaks

Overload Particle size (medium)

Particle size distribution

Post column derivatization

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SSJCP, Department of Pharmaceutical Analysis

SSJCP, Department of Pharmaceutical Analysis

SSJCP, Department of Pharmaceutical Analysis

Internal diameter of the column

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The Most Basic Explanation of Chromatography Ever

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Faster separation times

How Do You Get Separation? Hardware: pumps, injector, detector

Column: particle diameter, column size, packing materials

Column Considerations Theory (including, well...you know) Different Stationary Phases

Hardware Components Pumps, Injectors, Detectors, etc. Examples of Application-Specific Configurations

Modern HPLC v/s Traditional LC Methods

HPLC and Pre-HPLC Techniques

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MODES OF SEPARATION IN HPLC

Size exclusion chromatography

Based on mode of chromatography

Normal phase mode

Based on principle of separation

Based on elution technique

Based on the scale of operation

COLUMN TYPES Normal Phase LC

Nonpolar - mobile phase: Hexane, Ethyl acetate

Normal Phase HPLC Columns

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SSJCP, Department of Pharmaceutical Analysis

SSJCP, Department of Pharmaceutical Analysis

Rugged, general purpose, highly retentive

Moderate retention, normal & rev. phase

SSJCP, Department of Pharmaceutical Analysis