HPLC

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HPLC by RAVI PRATAP PULLA

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HPLC SSJCP, Department of Pharmaceutical Analysis 1 I/II, I st 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 .

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HPLC – THE DEVELOPMENT OF A NAME SSJCP, Department of Pharmaceutical Analysis 2 High PERFORMANCE Liquid Chromatography PRESSURE Price Prestige Peak Profit Propaganda Promise Philosophy Polite Problem Ph (F) antasy Pragmatic Pleasure Passion

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Introduction to Liquid Chromatography Columns System Components Applications Troubleshooting 3 SSJCP, Department of Pharmaceutical Analysis

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A Brief History of Chromatography  1903: Russian botanist Mikhail Tswett separated plant pigments  1 938 : 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. SSJCP, Department of Pharmaceutical Analysis 4

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 1966: HPLC was first named by Horvath at Yale University but HPLC didn’t “catch on” until the 1970s  1978: W.C. Stills introduced “flash chromatography”, where solvent is forced through a packed column with positive pressure. SSJCP, Department of Pharmaceutical Analysis 5

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Modern HPLC  Late 1970s/early 1980s ► Instrumentation developed for high pressure solvent delivery: pumps, autosamplers , diode array detectors ► More uniform packing material produced for columns  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. SSJCP, Department of Pharmaceutical Analysis 6

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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. SSJCP, Department of Pharmaceutical Analysis 7

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CHROMATOGRAPHY IS INCOMPLETE WITHOUT LEARNING FEW BASIC TERMINOLOGIES SSJCP, Department of Pharmaceutical Analysis 8 For any further clarification or details of the below content(s) feel free to mail me : ravipratappulla@gmail.com

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SSJCP, Department of Pharmaceutical Analysis 9 Absorption Bonded phase Additive Breakthrough volume Adsorbent Capillary column Adsorption Capillary LC Adsorption isotherm Cartridge column Affinity chromatography Cation exchange chromatography Agarose Channeling Alumina Chemisorption Amphoteric ion-exchange phase Chiral stationary phase Analyte Chlorosilane Anion exchange chromatography Co-ion Bed volume Column back pressure BET ( Brunauer , Emmet & Teller) method Column chromatography

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SSJCP, Department of Pharmaceutical Analysis 10 Column plate number Eluite Column switching Elute Column volume Elution Competing base Exclusion chromatography (Size) Counterion Extra column effects Coverage Fast protein LC (FPLC) Cross-links Frontal chromatography Degassing Gel filtration chromatography (GFC) Displacement chromatography Gradient elution Dynamic coating Graphitized carbon packing Effluent Guard column Eluate Heart cutting Eluent Hold-up volume ( V M or t M ) Dead time (t o / t m )

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SSJCP, Department of Pharmaceutical Analysis 11 Hydrophobic interaction chromatography (HIC) Ion exclusion Ion chromatography Ion moderated partioning chromatography (IMPC) Imprinted phases Ion pair chromatography (IPC) Indirect detection Linear chromatography Injector (sample) Linear velocity Inlet Liquid chromatography In-line filter Mobile phase velocity Interparticle porosity ( ee ) Open tubular column Interstitial volume Partition chromatography Intraparticle porosity ( ei ) Packed column Intraparticle volume Peak Ion exchange chromatography Peak area Ion chromatography (IC) Peak maximum

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SSJCP, Department of Pharmaceutical Analysis 12 Peak width Retention factor (k) Phase ratio Retention volume (V R or t R ) Plate height (H) Separation factor (a) Plate number (N) Solid support Pressure drop Solute Reduced mobile phase velocity (n) Stationary phase Resolution (Peak) [ R s ]/ Resolution(R) Tailing Reduced plate height (h) Void volume Relative Retention time (RRT) Retention time ( t R ) Interparticle time ( t Z ) Capacity factor (k’) Selectivity factor ( α ) Dead Volume( V d )

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SSJCP, Department of Pharmaceutical Analysis 13 Activity Adsorption chromatography Asymmetry Back pressure Back flushing Band spacing Baseline Baseline noise Baseline resolved peak Breakthrough volume Buffer Calibration standard Capacity factor Chain length Channeling Chromatogram Chromatographic conditions Chromatographic resolution Chromatographic system Column performance Dead volume ( V m ) Dead time (t m ) Detection Detector Detection threshold Detector linearity

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SSJCP, Department of Pharmaceutical Analysis 14 Detector sensitivity Differential Refractive Index( RI) Electrochemical detector Elution order Elution chromatography Eluotropic sequence Elution volume Extra column volume External standard Flow rate Fluorescence detector Frit Fronting HETP Hydrophilic Hydrophobic Internal standard Integrator Interstitial particle volume Ion exchanger Ion suppression Isocratic analysis Isothermal chromatography Ligand Loading matrix

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SSJCP, Department of Pharmaceutical Analysis 15 Organic modifier Overload Partially resolved peaks Particle size (medium) Particle size distribution Peak broadening Peak area Peak base Peak height Peak identification Peak Quantitation Peak shape Phase system Polarity Pore diameter Pore volume Post column derivatization Pre column Pulsating flow Recycling Regeneration Retention Retention time Retention volume Sample Sample capacity

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SSJCP, Department of Pharmaceutical Analysis 16 Sample preparation Separation capacity silanization Silanol groups Sorbent S.P chemically bonded S.P Surface modification Specific surface SFC( supercritical fluid chromatography) Vacancy chromatogram Void Void time

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IUPAC RECOMMENDATIONS & FREQUENTLY USED SYMBOLS IN CHROMATOGRAPHY SSJCP, Department of Pharmaceutical Analysis 17 PARAMETER SYMBOL Separation factor α Selectivity factor (up to 1993 A.D) α Area a / A Diameter d e Diffusion coefficient d Porosity ε / ε t Flow rate (volumetric) f Plate height h Viscosity η Equilibrium distribution constant k Rate constant k Retention factor k

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SSJCP, Department of Pharmaceutical Analysis 18 PARAMETER SYMBOL Capacity factor k’ Length of the column l / L Plate number /number of theoretical plates n / N Density ρ Pressure p / P Pressure (relative) p Radius r Temperature (absolute) t / T Time t Retention time t r / t R Velocity (linear) u Volume v

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SSJCP, Department of Pharmaceutical Analysis 19 PARAMETER SYMBOL Retention volume v r Mass (Weight) w Peak width w Difference ∆ Partial diameter d p Flow F Height equivalent of a theoretical plate(HETP) H Internal diameter of the column I.D Wavelength λ Iso electric point pK a Resolution R Death time t m / t 0

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SSJCP, Department of Pharmaceutical Analysis 20 PARAMETER SYMBOL Gradient time t G Net retention time t R ' Linear velocity μ Dead volume of apparatus V d Pore volume V p For any further clarification or details of the above content(s) feel free to mail me : ravipratappulla@gmail.com

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SSJCP, Department of Pharmaceutical Analysis 21 The Most Basic Explanation of Chromatography Ever

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 D rugs 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. SSJCP, Department of Pharmaceutical Analysis 22

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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. SSJCP, Department of Pharmaceutical Analysis 23

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SSJCP, Department of Pharmaceutical Analysis 24  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 SSJCP, Department of Pharmaceutical Analysis 25

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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. SSJCP, Department of Pharmaceutical Analysis 26

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HPLC and Pre-HPLC Techniques SSJCP, Department of Pharmaceutical Analysis 27

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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 SSJCP, Department of Pharmaceutical Analysis 28

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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 SSJCP, Department of Pharmaceutical Analysis 29

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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 SSJCP, Department of Pharmaceutical Analysis 30

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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. SSJCP, Department of Pharmaceutical Analysis 31

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SSJCP, Department of Pharmaceutical Analysis 32 Normal Phase HPLC Columns Cyano : `Rugged, moderate polarity, general use -OH ( Diol ) : More polar and retentive Amino : Highly polar, less stable Silica : Very rugged, low cost, adsorbent & Unbonded 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 SSJCP, Department of Pharmaceutical Analysis 33

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 more polar solvents in the mobile phase will decrease the retention time of analytes  hydrophobic solvents tend to induce slower eluti on (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 almost exclusively by an adsorptive mechanis m SSJCP, Department of Pharmaceutical Analysis 34

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Reversed-Phase LC  Nonpolar - stationary phase: C 8 , C 18  Polar - mobile phase: Water, ACN, Methanol  The MOST polar compound comes out first  Generally used for separation of polar compounds SSJCP, Department of Pharmaceutical Analysis 35

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RP-HPLC Columns C 18 , C 8 : Rugged, general purpose, highly retentive C 3 , C 4 : Less retentive, used mostly for peptides & proteins Phenyl : Greater selectivity than alkyl-bonded Cyano : Moderate retention, normal & rev. phase Amino : Weak retention, good for carbohydrates NOTE : The cyano column with a high polarity mobile phase (Water/ MeOH ) will act as a RP- Column. SSJCP, Department of Pharmaceutical Analysis 36

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 stationary phase is a silica which has been surface-modified with RMe 2 SiCl, where R is a straight chain alkyl group such as C 18 H 37  or C 8 H 17 .  retention time is longer for molecules which are less polar , while polar molecules elute more readily   can increase retention times by adding more w ater to the mobile phase  the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase SSJCP, Department of Pharmaceutical Analysis 37

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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 proportiona l to the contact surface area around the non-polar segment of the analyte molecule upon association with the ligand on the stationary phase . SSJCP, Department of Pharmaceutical Analysis 38

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SSJCP, Department of Pharmaceutical Analysis 39  solvophobic  effect is dominated by the force of water for "cavity-reduction" around the analyte and the C 18 -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, -NH 2 , COO –  or -NH 3 +  in their structure) are less retained as they are better i ntegrated into water.  interactions are subject to steric effect s 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. SSJCP, Department of Pharmaceutical Analysis 40

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 surface hindran ce typically results in less retention.  Retention time i ncreases 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. SSJCP, Department of Pharmaceutical Analysis 41

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SSJCP, Department of Pharmaceutical Analysis 42  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.  SSJCP, Department of Pharmaceutical Analysis 43

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 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.  C an 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 . SSJCP, Department of Pharmaceutical Analysis 44

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TYPICAL COLUMN SIZES SSJCP, Department of Pharmaceutical Analysis 45

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SSJCP, Department of Pharmaceutical Analysis 46

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 Particle size: 5 µm, 3 µm, 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 m 2 /g ► Carbon load: 9-12% for C 8 , 16-20% for C 18  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 47

RP-HPLC MECHANISM:

RP-HPLC MECHANISM  Synthesis of RP Packing  RP Column Properties  RP Retention Mechanisms  Important RP parameters  RP Optimization 48 SSJCP, Department of Pharmaceutical Analysis

Synthesis of RP Packing:

Synthesis of RP Packing 49 SSJCP, Department of Pharmaceutical Analysis

RP COLUMN PREPARATION:

RP COLUMN PREPARATION 50 SSJCP, Department of Pharmaceutical Analysis

COMMON RP PACKING:

COMMON RP PACKING 51 SSJCP, Department of Pharmaceutical Analysis

RP COLUMN PROPERTIES:

RP COLUMN PROPERTIES ► Hydrophobic Surface ► Particle Size and Shape ► Particle Size Distribution ► Porosity, Pore Size and Surface Area 52 SSJCP, Department of Pharmaceutical Analysis

PARTICLE SIZE:

PARTICLE SIZE ► Columns have a distribution of particle sizes ► Reported “particle diameter” is an average ► Broader distribution ---> broader peaks 53 SSJCP, Department of Pharmaceutical Analysis

Particle Size Distribution of several column batches:

Particle Size Distribution of several column batches Copyrights: Neue, HPLC Columns Theory, Technology and Practice , Wiley, 1997, p.82 54 SSJCP, Department of Pharmaceutical Analysis

RP MECHANISM (SIMPLE):

RP MECHANISM (SIMPLE) 55 SSJCP, Department of Pharmaceutical Analysis

RP Mechanism (Advanced):

RP Mechanism (Advanced)  Classical measures of retention ► capacity factors ► partition coefficients ► Van’t Hoff Plots  Give bulk properties only ► do not give molecular view of separation process 56 SSJCP, Department of Pharmaceutical Analysis

PROPOSED RP MECHANISMS:

PROPOSED RP MECHANISMS ► Hydrophobic Theory ► Partition Theory ► Adsorption Theory 57 SSJCP, Department of Pharmaceutical Analysis

HYDROPHOBIC THEORY:

HYDROPHOBIC THEORY  Chromatography of “cavities” in solvent created by hydrophobic portion of analyte molecule  Surface Tension  Interaction of polar functions with solvent  Stationary phase is passive 58 SSJCP, Department of Pharmaceutical Analysis

PARTITION THEORY:

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 SSJCP, Department of Pharmaceutical Analysis

ADSORPTION THEORY:

ADSORPTION THEORY  Analytes “land” on surface - do not penetrate  Non-polar interactions between analyte hydrophobic portion and bonded phase  Weak interactions ► dipole-dipole ► dipole-induced dipole ► induced dipole-induced dipole 60 SSJCP, Department of Pharmaceutical Analysis

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None of the above can completely explain all of the observed retention in RP-HPLC 61 SSJCP, Department of Pharmaceutical Analysis

IMPORTANT REVERSED PHASE PARAMETERS:

IMPORTANT REVERSED PHASE PARAMETERS  Solvent (mobile phase ) Strength  Choice of Solvent  Mobile Phase pH  Silanol Activity 62 SSJCP, Department of Pharmaceutical Analysis

SOLVENT STRENGTH:

SOLVENT STRENGTH  Water is “weak” solvent  Increased organic ---> decreased retention  Organic must be miscible with water 63 SSJCP, Department of Pharmaceutical Analysis

EFFECT OF SOLVENT:

EFFECT OF SOLVENT 64 SSJCP, Department of Pharmaceutical Analysis

SOLVENT STRENGTH:

SOLVENT STRENGTH COPYRIGHTS: Snyder and Kirkland, Introduction to Modern Liquid Chromatography , Wiley, 1979, p. 286. 65 SSJCP, Department of Pharmaceutical Analysis

VARYING SELECTIVITY:

VARYING SELECTIVITY COPYRIGHTS: Snyder and Kirkland, introduction to Modern Liquid Chromatography, Wiley, 1979, p. 287. 30% MeCN 70% Water 45% MeOH 55% Water 30x0.46 cm C-18, 1.5 mL.min,254 nm, 10 mg each 66 SSJCP, Department of Pharmaceutical Analysis

pH:

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 67 SSJCP, Department of Pharmaceutical Analysis

pH Effect on Retention:

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 . 68 SSJCP, Department of Pharmaceutical Analysis

Use of Buffers:

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 69 SSJCP, Department of Pharmaceutical Analysis

Common buffers:

Common buffers Useful buffering between pH 2-8. 70 SSJCP, Department of Pharmaceutical Analysis

Silanol Activity:

Silanol Activity  RP ligands occupy about 50% of silanols  Others are “active”  Weak acids 71 SSJCP, Department of Pharmaceutical Analysis

Silica Surface:

Silica Surface 72 SSJCP, Department of Pharmaceutical Analysis

Dealing with Residual Silanols:

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 73 SSJCP, Department of Pharmaceutical Analysis

Silanol Interactions:

Silanol Interactions  Hydrogen bonding  Dipole-dipole  Ion exchange  Low pH --> silanols protonated  Add basic modifier (TEA) to compete for sties 74 SSJCP, Department of Pharmaceutical Analysis

pH Effect on Tailing:

pH Effect on Tailing Neue, p196 75 SSJCP, Department of Pharmaceutical Analysis

RP Optimization:

RP Optimization 76 SSJCP, Department of Pharmaceutical Analysis

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77 SSJCP, Department of Pharmaceutical Analysis

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IDEALIZED HPLC SEPARATION SSJCP, Department of Pharmaceutical Analysis 78

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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 µm particles V m ≈ 0.5d c 2 L Where, V m is the column volume in mL, L is the column length in cm, and d c is the inner diameter in cm SSJCP, Department of Pharmaceutical Analysis 79

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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 SSJCP, Department of Pharmaceutical Analysis 80

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FACTORS INFLUENCING RESOLUTION  Capacity Factor, k’  Selectivity Factor, α  Efficiency, N SSJCP, Department of Pharmaceutical Analysis 81

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RESOLUTION  For closely eluting or adjacent peaks , the resolution equation may be expressed as:  The terms of capacity factor (k ’ ), selectivity ( α ), and efficiency (N) all contribute to resolution SSJCP, Department of Pharmaceutical Analysis 82

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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: R s = 2( V r b - V r a )/( W a + W b ) = 2( t r b - t r a )/ ( W a + W b ) Where, V r a /b = retention volume of peak a/b t r a /b = retention time of peak a/b W a /b = width of peak a/b SSJCP, Department of Pharmaceutical Analysis 83

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CAPACITY FACTOR, k ’  The relative degree to which an analyte component is delayed as it is eluted through a given system (retentivity).  The larger the k’, the later the analyte elutes after the void. SSJCP, Department of Pharmaceutical Analysis 84 k’ = (V r - V 0 )/V 0 = (t r - t 0 )/t 0 Where, V r = peak retention volume V 0 = column void volume t r = peak retention time t 0 = 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. SSJCP, Department of Pharmaceutical Analysis 85

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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. SSJCP, Department of Pharmaceutical Analysis 86

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Mobile Phase Strength v/s k ’ SSJCP, Department of Pharmaceutical Analysis 87 4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water 100% 90% 80% 70% 60% 50% 0.079 0.212 0.472 1.127 2.813 Capacity Factor for Butyl Paraben (Peak 4) 7.666 100% 90% 80% 70% 60% 50%

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Temperature Effect on k ’ SSJCP, Department of Pharmaceutical Analysis 88 20°C 25°C 30°C 35°C 40°C 45°C 50°C 2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH/Water

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Summary of k ’ 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. SSJCP, Department of Pharmaceutical Analysis 89

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Selectivity Factor, α  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: α = k’b / k’a  If α = 1, two components are perfectly overlapping  For early eluting peaks you want α to be large for good resolution.  For later eluting peaks, α can be smaller and still have acceptable separation. SSJCP, Department of Pharmaceutical Analysis 90

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Effect of α on Overall Resolution  Remember the resolution equation?  Let’s only look at the part involving α  And see how much resolution will improve with small changes in α SSJCP, Department of Pharmaceutical Analysis 91

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 For an α value of 1.1, the contribution of the selectivity term is (1.1 – 1) / 1.1 = 0.09  For an α value of 1.4, the contribution of the selectivity term is (1.4 – 1) / 1.4 = 0.29  So, a very small change in α leads to a more than THREE-FOLD increase in the contribution to resolution. SSJCP, Department of Pharmaceutical Analysis 92

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 As α grows larger, its effect reaches a limit at a value of about 5.  Since α depends on components’ retention factor k ’ , longer columns eventually have a diminished effect on resolution. SSJCP, Department of Pharmaceutical Analysis 93

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Influencing the Selectivity Factor α  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 94

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Summary of α Effects  Since α 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 ( α ) may affect resolution ► Increasing the temperature decreases individual values of k ’ , but their ratio ( α ) may significantly affect resolution.  A small increase in α leads to a large increase in resolution SSJCP, Department of Pharmaceutical Analysis 95

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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 numb er. SSJCP, Department of Pharmaceutical Analysis 96

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Calculation of Theoretical Plates N = A( t r /W) 2 W A Method Width measured at W i 4 Inflection point (60.7% of peak height) W h 5.54 ½ Height 50% of peak height W 3s 9 3s 32.4% of peak height W 4s 16 4s 13.4% of peak height W 5s 25 5s 4.4% of peak height W b 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. SSJCP, Department of Pharmaceutical Analysis 97

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Calculation of Efficiency, N SSJCP, Department of Pharmaceutical Analysis 98 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?  Now let’s look at the part involving N  And see how much resolution will improve with changes in N SSJCP, Department of Pharmaceutical Analysis 99

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 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 SSJCP, Department of Pharmaceutical Analysis 100 PLATE √ N CONTRIBUTION 5000 70.7 ----- 10,000 100 41% 20,000 141.4 100%

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Effect of N on Overall Resolution  Note that there is no flattening of the curve like with k ’ and α .  Resolution will continue to increase as theoretical plates increase. SSJCP, Department of Pharmaceutical Analysis 101

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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 102

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Effect of Particle Size on N  Smaller particle sizes result in higher numbers of theoretical plates SSJCP, Department of Pharmaceutical Analysis 103

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Relative Influence of All Factors on Resolution Note that changing α a very small amount has the biggest effect SSJCP, Department of Pharmaceutical Analysis 104 Parameter Change N k’ α R s Standard 10,000 2 1.1 1.52 +10% N 11,000 2 1.1 1.59 -25% N 7,500 2 1.1 1.31 -50% N 5,000 2 1.1 1.07 -60% N 4,000 2 1.1 0.96 -75% N 2,500 2 1.1 0.76 +10% k’ 10,000 2.2 1.1 1.56 +10% α 10,000 2 1.2 2.78

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Review of Factors SSJCP, Department of Pharmaceutical Analysis 105 PARAMTER INFLUENCED BY TARGET VALUE Efficiency, N Column, system flow path, configuration Minimum of 400 theoretical plates /cm Capacity factor, k’ MP strength 1.0 - 10 Selectivity, α M.P & S.P type 1.1 - 2 Resolution, R s All of the above 1.3 – 1.5 or greater

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Normal Phase v/s Reversed Phase SSJCP, Department of Pharmaceutical Analysis 106 PARAMETER NP RP Polarity of packing Medium to high Low to medium Polarity of solvent Low to medium Medium to high Elution sequence Low polarity first High polarity first Increase solvent polarity Faster elution Slower elution

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ION EXCHANGE CHROMATOGRAPHY SSJCP, Department of Pharmaceutical Analysis 107

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 based on the a ttraction between solute ions and charged sites bound to the stationary phase.  The stationary phase contains ionic groups like NR⁺ з , SO⁻ з 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 cha rge 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

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 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  SSJCP, Department of Pharmaceutical Analysis 109

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 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. SSJCP, Department of Pharmaceutical Analysis 110

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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 SSJCP, Department of Pharmaceutical Analysis 111

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Examples  Stationary phase contains charged groups  SAX (Strong Anion Exchange): NH 3 +  WAX (Weak Anion Exchange): NR 2 H + (DEAE) [ D i E thyl A mino E thanol]  SCX (Strong Cation Exchange): SO 3 -  WCX (Weak Cation Exchange): C arboxy M ethyl (CM)  More highly charged analytes have stronger retention  More “bulky” stationary phases have weaker retention SSJCP, Department of Pharmaceutical Analysis 112

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 I EC 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 SSJCP, Department of Pharmaceutical Analysis 113

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AFFINITY/ BIOAFFINITY CHROMATOGRAPHY SSJCP, Department of Pharmaceutical Analysis 114

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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. SSJCP, Department of Pharmaceutical Analysis 115

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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. SSJCP, Department of Pharmaceutical Analysis 116

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Size Exclusion LC (or) Gel Permeation (or) Gel filtration SSJCP, Department of Pharmaceutical Analysis 117

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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   and quaternary 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. SSJCP, Department of Pharmaceutical Analysis 118

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 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   ( R H ).  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 purificati on. SSJCP, Department of Pharmaceutical Analysis 119

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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 organic-soluble 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. SSJCP, Department of Pharmaceutical Analysis 120

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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. SSJCP, Department of Pharmaceutical Analysis 121

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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. SSJCP, Department of Pharmaceutical Analysis 122

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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 123

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 Gradient elution decreases the retention of the later-eluting 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. SSJCP, Department of Pharmaceutical Analysis 124

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 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 phas e  is to reduce this high order and thus reduce the retarding strength of the aqueous component. SSJCP, Department of Pharmaceutical Analysis 125

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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 SSJCP, Department of Pharmaceutical Analysis 126

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ISOCRATIC SEPARATION  1 pump and premixing  4.6 mm ID Column, 1 mL/min, Changing MeOH % vs Water SSJCP, Department of Pharmaceutical Analysis 127

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SSJCP, Department of Pharmaceutical Analysis 128  1 pump with valve and premixing A = 80% Methanol, 20% Water B = 70% Methanol, 30% Water C = 60% Methanol, 40% Water D = 50% Methanol, 50% Water To Column A B C D

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 1 pump with mixer – let the pump do the work! Method 1: A.CONC = 20%, B.CONC = 80% Method 2: A.CONC = 30%, B.CONC = 70% Method 3: A.CONC = 40%, B.CONC = 60% Method 4: A.CONC = 50%, B.CONC = 50% SSJCP, Department of Pharmaceutical Analysis 129 To Column A B C D

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LOW PRESSURE GRADIENT 1 Pump, solvents are mixed before the pump Requires degassing SSJCP, Department of Pharmaceutical Analysis 130 To Column A B C D

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HIGH PRESSURE GRADIENT Binary Gradient 2 Pumps and Mixer SSJCP, Department of Pharmaceutical Analysis 131 ………. ………. ………. ………. ………. ………. Ternary Gradient 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 SSJCP, Department of Pharmaceutical Analysis 132

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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 133

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PRINCIPLE OF SEPARATION  The principle of separation is A dsorption.  Separation of components takes place because of the difference in affinity of compounds towards stationary phase. SSJCP, Department of Pharmaceutical Analysis 134

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SSJCP, Department of Pharmaceutical Analysis 135  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. 2 1 Stronger interaction Weaker interaction

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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 today’s 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. SSJCP, Department of Pharmaceutical Analysis 136

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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. SSJCP, Department of Pharmaceutical Analysis 137

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INSTRUMENTATION SSJCP, Department of Pharmaceutical Analysis 138

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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. High-pressure 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 SSJCP, Department of Pharmaceutical Analysis 139

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BASIC FLOW CHART OF A HPLC SYSTEM SETUP SSJCP, Department of Pharmaceutical Analysis 140

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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 SSJCP, Department of Pharmaceutical Analysis 141

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 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 SSJCP, Department of Pharmaceutical Analysis 142

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 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 SSJCP, Department of Pharmaceutical Analysis 143

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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. SSJCP, Department of Pharmaceutical Analysis 144

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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 SSJCP, Department of Pharmaceutical Analysis 145

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Various solvent delivery systems SSJCP, Department of Pharmaceutical Analysis 146

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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 SSJCP, Department of Pharmaceutical Analysis 147

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 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 SSJCP, Department of Pharmaceutical Analysis 148

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SSJCP, Department of Pharmaceutical Analysis 149  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 (35-400µl), 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.

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HPLC PUMPS – TWO BASIC TYPES  Tandem piston ► Two pistons with different volumes (48 and 24 µL) ► During each stroke, 24 µL 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 SSJCP, Department of Pharmaceutical Analysis 150

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TANDEM PISTON PUMP SSJCP, Department of Pharmaceutical Analysis 151 ← Primary Piston Secondary Piston ↓

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DUAL PISTON  Two pistons with equal volume (10 µL each)  During each stroke, 10 µL is delivered  Best for low flow rates (< 1 mL/min)  Little to NO pulsation  S o it’s ideal for pulse sensitive detectors like RID and CDD SSJCP, Department of Pharmaceutical Analysis 152

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DUAL PISTON SSJCP, Department of Pharmaceutical Analysis 153

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SSJCP, Department of Pharmaceutical Analysis 154 OTHER PUMP COMPONENTS  Check Valves ► Control liquid movement in and out of the pump head

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 Piston/plunger seal ► Prevents solvent leakage out of pump head  Inline filter ► Removes solvent particulates SSJCP, Department of Pharmaceutical Analysis 155

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VALVES USED WITH PUMPS  Solvent Selection – 2 Solvents Per Pump ► Use for solvent switching SSJCP, Department of Pharmaceutical Analysis 156 A B C 1 2 ………. ………. ………. A ………. ………. ………. C ………. ………. ………. B 1 2 2 1

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 Solvent Selection – 2 Solvents Per Pump ► Use for pump loading of large sample volumes SSJCP, Department of Pharmaceutical Analysis 157 Pump A – weak gradient solvent and sample loading A B C 1 2 Sample Weak gradient solvent Pump B – strong gradient solvent. Form the gradient with B.CONC command

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 Solvent Selection – 4 Solvents Per Pump ► Use for low pressure gradient formation SSJCP, Department of Pharmaceutical Analysis 158 To Column Combine any proportion of A/B/C/D. REQUIRES additional mixing before the injector. A B C D

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 Solvent Selection – 4 Solvents Per Pump ► Use for different gradients in method development SSJCP, Department of Pharmaceutical Analysis 159 A B Pump A Pump B A A B B C C D D 4 Pairs A B Pump A Pump B A A , B , C , D B A , B , C , D C A , B , C , D D A , B , C , D 16 Combinations

SAMPLE INJECTION SYSTEM:

SAMPLE INJECTION SYSTEM 160  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. SSJCP, Department of Pharmaceutical Analysis

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161 SSJCP, Department of Pharmaceutical Analysis

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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 SSJCP, Department of Pharmaceutical Analysis 162

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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 163

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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 SSJCP, Department of Pharmaceutical Analysis 164

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SAMPLE INJECTION – FIXED LOOP  External syringe draws sample, then fills the fixed-volume loop attached to the valve . SSJCP, Department of Pharmaceutical Analysis 165

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 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 SSJCP, Department of Pharmaceutical Analysis 166

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SAMPLE INJECTION TO FLOW PATH SSJCP, Department of Pharmaceutical Analysis 167 Sample Injection – Everything drawn into the needle goes to the column. Sample Loading

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RINSING AFTER INJECTION SSJCP, Department of Pharmaceutical Analysis 168 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 MUST be degassed!

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169 Varian 9010 Solvent Delivery System Rheodyne Injector %A %B %C Flow Rate Pressure {H 2 O} {MeOH} (mL/min) (atmos.) Ready Ternary Pump A C B from solvent reservoir Column to detector to column through pulse dampener to injector through pump load inject SSJCP, Department of Pharmaceutical Analysis

CHROMATOGRAPHIC COLUMN:

CHROMATOGRAPHIC COLUMN 170  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 25µm 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 SSJCP, Department of Pharmaceutical Analysis

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 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 µm)  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 solvents) SSJCP, Department of Pharmaceutical Analysis 171

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HPLC Columns Packing Materials  Columns are packed with small diameter porous particles.  The most popular sizes are: 5-μm, 3.5- μm and 1.8-μm  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, C 18 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 col umn. SSJCP, Department of Pharmaceutical Analysis 172

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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 SSJCP, Department of Pharmaceutical Analysis 173

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Why Use a Column Oven ?  Retention times decrease & higher flow rates possible SSJCP, Department of Pharmaceutical Analysis 174 20°C 25°C 30°C 35°C 40°C 45°C 50°C 2.1 mm ID Column, 0.35 mL/min, 50/50 MeOH /Water

DETECTORS:

DETECTORS 175  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. 171 SSJCP, Department of Pharmaceutical Analysis 171

TYPES OF DETECTORS:

TYPES OF DETECTORS 176 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

PHOTOMETRIC DETECTORS:

PHOTOMETRIC DETECTORS 177  These normally operate in the ultra violet region of the spectrum .  Most extensively used in pharmaceutical analysis. SSJCP, Department of Pharmaceutical Analysis

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178 SINGLE WAVELENGTH DETECTORS  Equipped with a low pressure mercury discharge lamp.  The absorbance is measured at the wavelength of mercury at 254 nm. SSJCP, Department of Pharmaceutical Analysis

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179 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. SSJCP, Department of Pharmaceutical Analysis

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Multi-wavelength UV-Vis Absorption Detector Deuterium Lamp Photodiode Array 180 SSJCP, Department of Pharmaceutical Analysis

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181 VARIABLE WAVELENGTH DETECTORS  Use a deuterium light source.  A grating monochromator to allow selection of any wavelength in deuterium continuum (190-360 nm). SSJCP, Department of Pharmaceutical Analysis 177

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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 SSJCP, Department of Pharmaceutical Analysis 182

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SSJCP, Department of Pharmaceutical Analysis 183 UV-Visible Detector

Variable wavelength detector:

Variable wavelength detector 184 SSJCP, Department of Pharmaceutical Analysis

Variable UV/Vis Detector:

Variable UV/Vis Detector 185 ABS AUFS l RunTime EndTime 0.001 2.000 238 0.00 min 10.0 min Ready SSJCP, Department of Pharmaceutical Analysis

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186 PROGRAMMABLE DETECTORS  Allow the automatic change of wavelength between and during the chromatographic analysis. SSJCP, Department of Pharmaceutical Analysis

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187 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. SSJCP, Department of Pharmaceutical Analysis 183

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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 SSJCP, Department of Pharmaceutical Analysis 188

DIODE ARRAY DETECTOR:

DIODE ARRAY DETECTOR 189 SSJCP, Department of Pharmaceutical Analysis

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190 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. SSJCP, Department of Pharmaceutical Analysis

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 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) SSJCP, Department of Pharmaceutical Analysis 191

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Fluorescence Detector 192 SSJCP, Department of Pharmaceutical Analysis

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193 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. SSJCP, Department of Pharmaceutical Analysis

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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 194

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RI BALANCE  Fill sample and reference cell with mobile phase SSJCP, Department of Pharmaceutical Analysis 195

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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. SSJCP, Departme nt of Pharmaceutical Analysis 196

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Refractive Index Detector 197 SSJCP, Department of Pharmaceutical Analysis

ELECTROCHEMICAL DETECTORS:

ELECTROCHEMICAL DETECTORS 198  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. SSJCP, Department of Pharmaceutical Analysis

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Electrochemical Detector 199 SSJCP, Department of Pharmaceutical Analysis

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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 temperatures SSJCP, Department of Pharmaceutical Analysis 200

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ELSD OPERATION SSJCP, Department of Pharmaceutical Analysis 201 Light Source PMT Amplifier Light Scattering Cell Nebulizer Nebulizer Gas (Air or Nitrogen) Drift Tube (Heated Zone Evaporation Area) Column Effluent Signal Output Analyte Nebulization Chamber

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ELSD v/s OTHER DETECTORS  ELSD has higher sensitivity than UV and RID  ELSD can be used with gradients, unlike RID SSJCP, Department of Pharmaceutical Analysis 202

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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 SSJCP, Department of Pharmaceutical Analysis 203

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 Use in Environmental and water testing ► Fl - , Cl - NO 3 - , PO 4 3- , SO 4 2- ► Li + , Na + , K + , Mg 2+ , Cu 2+ , 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 204

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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 205

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TWO IONIZIZATION TYPES  APCI: Atmospheric Pressure Chemical Ionization ► 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 SSJCP, Department of Pharmaceutical Analysis 206

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MS DETECTOR SSJCP, Department of Pharmaceutical Analysis 207 Orthogonal source geometry Heated capillary Q-array Octapole Quadrupole mass analyser Electron Multiplier Detector

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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 SSJCP, Department of Pharmaceutical Analysis 208

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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 4°C SSJCP, Department of Pharmaceutical Analysis 209

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How is Temperature Control Achieved?  Three (3) ways the temperature of a column could be controlled, use: ► Oven ► Heater Block ► Water bath SSJCP, Department of Pharmaceutical Analysis 210

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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. SSJCP, Department of Pharmaceutical Analysis 211

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 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…. SSJCP, Department of Pharmaceutical Analysis 212

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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. SSJCP, Department of Pharmaceutical Analysis 213

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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. SSJCP, Department of Pharmaceutical Analysis 214

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Preparation of Pure Compound(s)  By collecting the chromatographic peaks at the exit of the detector  and concentrating the compound (analyte) by removing/evaporating the solvent  a pure substance can be prepared for later use (e.g. organic synthesis, clinical studies, toxicology studies, etc….).  This methodology is called preparative chromatography. SSJCP, Department of Pharmaceutical Analysis 215

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Trace analysis  A trace compound is a compound that is of interest to the analyst but it’s 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

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SSJCP, Department of Pharmaceutical Analysis 217

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SEPARATION TECHNIQUES IN HPLC METHOD DEVELOPMENT GOAL COMMENT Resolution Precise and rugged quantitative analysis requires that R s be greater than 1.5 Separation time 3-10 min is desirable for routine procedures Quantitation ≤2% for assays; ≤ 5% for less-demanding analyses; ≤ 15% for trace analyses Peak Height Narrow peaks are desirable for large signal/noise ratios Solvent composition Minimum mobile-phase use per run is desirable 218 SSJCP, Department of Pharmaceutical Analysis

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THE VALIDATION PROCESS ► It consists of four distinct steps:  Software validation  Hardware (instrumentation) validation/qualification  Method validation  System suitability 219 SSJCP, Department of Pharmaceutical Analysis

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HPLC SYSTEM QUALIFICATION 220 SSJCP, Department of Pharmaceutical Analysis

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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 221 SSJCP, Department of Pharmaceutical Analysis

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► 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. 222 SSJCP, Department of Pharmaceutical Analysis

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► 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 SSJCP, Department of Pharmaceutical Analysis

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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 SSJCP, Department of Pharmaceutical Analysis

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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 225 SSJCP, Department of Pharmaceutical Analysis

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► 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 226 SSJCP, Department of Pharmaceutical Analysis

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► 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 227 SSJCP, Department of Pharmaceutical Analysis

STEPS FOR HPLC METHOD DEVELOPMENT :

STEPS FOR HPLC METHOD DEVELOPMENT Information on sample, define separation goals Need for special procedure sample pretreatment, etc Choose detector and detector settings Choose LC method; preliminary run; estimate the best separation conditions Validate method for release to routine laboratory Quantitative calibration Check for problems or requirement for special procedure Optimize separation conditions 228 SSJCP, Department of Pharmaceutical Analysis

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PARAMETERS USED IN METHOD VALIDATION 229 SSJCP, Department of Pharmaceutical Analysis

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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 230 SSJCP, Department of Pharmaceutical Analysis

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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. 231 SSJCP, Department of Pharmaceutical Analysis

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► 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 SSJCP, Department of Pharmaceutical Analysis

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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 233 SSJCP, Department of Pharmaceutical Analysis

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► Report % recovery (mean result and RSD): 100±2% ► 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 SSJCP, Department of Pharmaceutical Analysis

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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 ● σ = the standard deviation of the response, based on the standard deviation of the blank & the calibration curve & S = Slope 235 SSJCP, Department of Pharmaceutical Analysis

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► 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 236 SSJCP, Department of Pharmaceutical Analysis

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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. 237 SSJCP, Department of Pharmaceutical Analysis

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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 SSJCP, Department of Pharmaceutical Analysis

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Center for Drug Evaluation and Research (CDER) Limits for SST 239 SSJCP, Department of Pharmaceutical Analysis

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CHARACTERISTICS TO BE VALIDATED IN HPLC SSJCP, Department of Pharmaceutical Analysis 240 CHARACTERISTICS ACCEPTANCE CRITERIA Accuracy/trueness Recovery 98-102% with 80, 100 & 120% spiked sample Repeatability RSD < 2% Intermediate precision RSD < 2% Specificity/selectivity No interference Detection limit S/N > 2 or 3 Quantitation limit S/N > 10 Linearity Correlation coefficient r > 0.999 Range 80 – 120% Stability of sample solution > 24 hours or > 12 hours

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TYPICAL HPLC INSTRUMENT VERIFICATION REPORT SSJCP, Department of Pharmaceutical Analysis 241 TEST ITEM USER LIMIT ACTUAL LIMIT DAD noise < 5 X 10 -5 AU 1 X 10 -5 AU Baseline drift < 2x 10 -3 AU/hour 1.5 X 10 -4 AU/hour DAD WL calibration ± 1 nm ± 1 nm DAD linearity 1.5 AU 2.2 AU Pump performance < 0.3% RSD RT 0.15% RSD RT Temperature stability (column heater) ± 0.15° C ± 0.15° C Precision of peak area 0.5% RSD 0.09% RSD

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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 SSJCP, Department of Pharmaceutical Analysis

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TROUBLE SHOOTING (TIPS & FACTS) SSJCP, Department of Pharmaceutical Analysis 243 For any further clarification or details of the below content(s) feel free to mail me : ravipratappulla@gmail.com ASK PULLA

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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? SSJCP, Department of Pharmaceutical Analysis 244

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SSJCP, Department of Pharmaceutical Analysis 245 SIMPLE TESTS & DECISION CRITERIA 10. What does the name of a column tell us? 11. Is this C 18 column the right choice for my sample? 12. Why are polar solutes well separated with one C 18 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? 20. Does the pump work correctly, precisely or accurately?

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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

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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? SSJCP, Department of Pharmaceutical Analysis 247

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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

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SITUATION/SYMPTOM/CAUSE EQULIBRATION 53. SLOW COLUMN  RP- Ion pairing long chain 54. VARYING / VARIABLE RETENTION TIMES  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

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 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

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SSJCP, Department of Pharmaceutical Analysis 251  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

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 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 252

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 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. SSJCP, Department of Pharmaceutical Analysis 253

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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 SSJCP, Department of Pharmaceutical Analysis 254

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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  detector’s linear range  sample losses during sample preparation  sample losses on column peak too broad SSJCP, Department of Pharmaceutical Analysis 255

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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 256

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 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 257

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 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 SSJCP, Department of Pharmaceutical Analysis 258

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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

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 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

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 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

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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 Analys is 262

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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 SSJCP, Department of Pharmaceutical Analysis 263

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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 264

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74. RETENTION BEYOND TOTAL PERMEATION VOLUMES  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  leaking pump check valve or seals, pump cavitations SSJCP, Department of Pharmaceutical Analysis 265

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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 266

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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 267

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 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 SSJCP, Department of Pharmaceutical Analysis 268

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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 SSJCP, Department of Pharmaceutical Analysis 269

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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 270

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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 271

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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 TAIL IN INITIAL AND LATER INJECTIONS  Sample reacting with active sites  Wrong M.P pH or column type or injection solvent  Small void at column inlet  Unswept dead volume SSJCP, Department of Pharmaceutical Analysis 272

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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 SSJCP, Department of Pharmaceutical Analysis 273

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SSJCP, Department of Pharmaceutical Analysis 274 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

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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

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 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 SSJCP, Department of Pharmaceutical Analysis 276

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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 SSJCP, Department of Pharmaceutical Analysis 277

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 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 SSJCP, Department of Pharmaceutical Analysis 278

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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. SSJCP, Department of Pharmaceutical Analysis 279

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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 SSJCP, Department of Pharmaceutical Analysis 280

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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

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 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 282

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 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 SSJCP, Department of Pharmaceutical Analysis 283

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100. PROBLEMS RELATED TO ASSAY TYPE ANALYSIS SSJCP, Department of Pharmaceutical Analysis 284 EFFECT SITUATION/SYMPTOM CAUSE 1. Poor peak height reproducibility With all peaks 1. Irreproducible sample volume 2. Improper detector or recorder response 3. Column or detector is sample overloaded 4. Calibration & sample solutions decomposing 5. Internal standard decomposing or reacting 6. Irreproducible RT 2. With symmetrical peaks With all peaks As all above (read cause 1) Sample decomposition in column

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SSJCP, Department of Pharmaceutical Analysis 285 EFFECT SITUATION/SYMPTOM CAUSE 3. With unsymmetrical peaks With all peaks As above (read cause 1) Undesired interaction of solute with S.P Poorly packed column Sample decomposition in column 4. Poor peak area reproducibility With all peaks As above (read cause 1 ) Improper integrator or computer response Internal standard decomposing With symmetrical peaks As above (read causes 1 & 2) With unsymmetrical peaks As above (read causes 1 & 3)

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SSJCP, Department of Pharmaceutical Analysis 286 EFFECT SITUATION/SYMPTOM CAUSE 5. Poor analytical accuracy Analytical results higher than theoretical Improperly made standards Calibration or sample solution decomposing Unknown interference with peaks for desired components Internal standard reacting or decomposing Component of interest or internal standard too close Analytical results lower than theoretical Improperly made standards Calibration and or sample solution decomposing Unknown interference with internal standard peak

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SSJCP, Department of Pharmaceutical Analysis 287 EFFECT SITUATION/SYMPTOM CAUSE 6. Peak height or peak area calibrations do not intercept zero No peak seen for very small sample Desired component decomposing Improper peak size measurement Strange peak seen at or near R.T of component of interest Peak overlap Peak seen in control or blank run Contaminated injector 7. Variable analytical accuracy as function of concentration Non linear calibration plot or calibration factor Non linear detector response Sample component or internal standard decomposing

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SSJCP, Department of Pharmaceutical Analysis 288 EFFECT SITUATION/SYMPTOM CAUSE 8. Analytical precision insufficient for need Analytical variability too large even though proper procedure followed Less precise calibration method used lower precision with internal standard Equipment deficiencies Gradient elution used 9. Analytical speed insufficient for needs Higher analysis speed required for routine analysis or process analysis Low column plate count Low column selectivity Resolution of components too good

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SSJCP, Department of Pharmaceutical Analysis 289 EFFECT SITUATION/SYMPTOM CAUSE 10. Difficult to determine which peak is to be measured Irreproducible sample profile, peak sequences or R.T Poor separation reproducibility Column irreproducibility 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)

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101. PROBLEMS RELATED TO TRACE ANALYSIS SSJCP, Department of Pharmaceutical Analysis 290 EFFECT SITUATION/SYMPTOM CAUSE 11. Insufficient sensitivity for needs Poor detectability for peaks of interest Insufficient detector response Improper detector Sample volume too small K’ too large Column too long Value for α too small N too small Separation method not optimized Poor detection when sample volume is limiting Column I.D is too large

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SSJCP, Department of Pharmaceutical Analysis 291 EFFECT SITUATION/SYMPTOM CAUSE 12. Poor trace analysis accuracy or precision Overlap of trace peak with interferences Use of peak area method Insufficient resolution Interference from spurious unknown peaks Poor recoveries with sample pretreatment Variable loss of components of interest Change in calibration factor As above (read cause 1) 13. Analysis time too long Higher analysis speed required for routine analysis or process analysis As above (read cause 9)

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SSJCP, Department of Pharmaceutical Analysis 292 EFFECT SITUATION/SYMPTOM CAUSE Base line drift makes measurement of peaks difficult Detector drift Solvent contamination Broad peaks from previous runs are eluting during the analysis Flow rate change Gradient used at higher detector sensitivity Column bleed 14. Analysis time too long As above (read situation 9) As above (read cause 9) Components eluting long after peak of interest Improper separation system Simple isocratic system used Too complex sample

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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

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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- H 3 PO 4 , CF 3 COOH & HClO 4 ? SSJCP, Department of Pharmaceutical Analysis 294

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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 295

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125. If we have certain concentration of phosphate buffer and then adjust with phosphoric acid how do we calculate the amount of H 2 PO 4 - 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 pk a 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

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COMMON IMPURITIES IN HPLC SOLVENTS SSJCP, Department of Pharmaceutical Analysis 297 CONTAMINATION SOURCE SYMPTOMS Particulate matter Unclean vessel 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 Water Suring solvent preparation or improperly dried glassware's Stability of bonded phases alcohols Behave like water Peroxides Degradation Reaction with sample, column packing

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SSJCP, Department of Pharmaceutical Analysis 298 Dissolved oxygen Solvent preparation Degradation BHT Antioxidant in THF UV absorbing Bacterial or fungal growth Prolonged storage Can block the in line filters, column frit and degradation of bonded phases Halogens Chlorinated solvents can form HBr & or HCl Attack stainless steel part of the system

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ANALYST QUOTES  No one is ever born talented, nor is talent lucky coincidence, it is acquired through one’s 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. SSJCP, Department of Pharmaceutical Analysis 299

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 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. SSJCP, Department of Pharmaceutical Analysis 300

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 An ordinary teacher tells, good teacher explains, superior teacher demonstrates and the great teacher inspires.  Very few people really appreciate other’s work. There are very rare people who enjoy other’s 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.  Don’t 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 won’t 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.  Don’t fall in love with your own work or skill to the exclusion f other good ideas. SSJCP, Department of Pharmaceutical Analysis 301

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 Thanks not those faithful who simply praise words and actions but those who kindly re-prove the fault.  One can’t 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 one’s 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. SSJCP, Department of Pharmaceutical Analysis 302

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 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.   SSJCP, Department of Pharmaceutical Analysis 303

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SSJCP, Department of Pharmaceutical Analysis 304

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