2. TYPES OF HPLC NORMALLY EMPLOYED
NORMAL
PHASE
SP-POLAR
MP-RELATIVELY
LESS POLAR
REVERSE
PHASE
SP-NON POLAR
MP-RELATIVELY
MORE POLAR
HYDROPHILLIC
INTERACTION
CHROMTOGRAPHY
(HILIC)
VARIANT OF
NP-HPLC
SP-POLAR
MP-ORGANIC
PHASE + WATER
(<20%)
HYDROPHOBIC
INTERACTION
CHROMATOGRAPHY
(HIC)
A TYPE OF RP-
HPLC
USED FOR
BIOMOLECULES
7. DEFINING THE METHOD
SAMPLE
CHARACTERISTICS
METHOD
IMPLICATIONS
• NATURE OF THE SAMPLE
• PHYSICO-CHEMICAL PROPERTIES
• SOLUBILITY
• pKa
• CHIRALITY
• SPECTRA-UV,IR,NMR,MS
• METABOLIC AND DEGRADATION
PATHWAYS
• METHOD DEVELOPMENT TYPE
• MAXIMUM RUN FOR ANALYSIS
• NO.OF SAMPLES IN ROUTINE
ANALYSIS
• COMPLEXITY OF THE MIXTURE
• NO.OF COMPONENTS IN THE
MIXTURE
8.
9. COMMON MISTAKES
Inadequate formulation of method goals
Insufficient knowledge of chemistry
Use of the first reversed phase HPLC column available
Use of wrong instrument set-up
Trial and error with different columns, mobile phases
10. METHOD DEVELOPMENT PROTOCOL
COLLECTING ANALYTE INFORMATION IN REGARD TO PHYSICO-CHEMICAL PROPERTIES
DETERMINING SUITABLE MODE OF DETECTION FOR ANALYSIS
SAMPLE PREPARATION
(EXTRACTION,CENTRIFUGATION, FILTRATION, SONICATION, TYPE OF DILUENT)
DETERMINATION OF SOLUTION STABILITY IN DILUENT
CHOICE OF MOBILE PHASE AND GRADIENT CONDITIONS (MOST CRUCIAL STEP)
STATIONARY PHASE SELECTION AND ITS STABILITY AT OPERATIONAL MODE OF MOBILE
PHASE pH
OPTIMIZAION OF SEPARATION CONDITIONS
TROUBLESHOOT THE PROBLEMS
VALIDATE THE DEVELOPED METHOD FOR ROUTINE ANALYSIS
11. SAMPLE PROPERTIES
• ANALYTE STRUCTURE AND
pKa
• IONOGENIC NATURE OF
ANALYTE
• IF ANALYTE IS NEUTRAL
• ELUENT pH WILL HAVE NO
EFFECT
• HOWEVER THE DEGRADATION
PRODUCTS MUST BE
CONSIDERED
• IF ANALYTE IS IONIZABLE
• pKa IS ESSENTIAL
• pKa PREDICTION &
DETERMINATION
pKa PREDICTION
• ACD LABS
• PALLAS
• CHEMICALIZE.ORG
• CHEMSPIDER.COM
• DRUGBANK
• CHEMIDPLUS
• PUBCHEM
• CHEMBLDB
EXPERIMENTAL
DETERMINATION OF pKa
• POTENTIOMETRIC
TITRATIONS
• OCTANOL-WATER
PARTITION RATIOS
• SOLUBILITY DATA
• SPECTROPHOTOMETRIC
METHODS
• NMR TITRATIONS
• CAPILLARY
ELECTROPHORESIS
• LC METHODS
• LC-CE
12. pKa of Some Common Functional Groups
aAddition of R(methyl, ethyl, etc.) group on aromatic ring or on NR2 will cause an increase of compound
pKa due to electron- donating effects from methyl groups.
bSubstitution in general of halogens on aromatic ring will decrease compound pKa. Example: o-
chloroaniline pKa = 2.6, aniline pKa = 4.6.
13. SOLUBILITY OF COMPONENTS AND DILUENT
EFFECTS (MATRIX EFFECTS)
• Solubility of the analyte is also very important
• Solubility of a particular drug is a prerequisite for any salt selection
program
• The free acid/free base and their corresponding salts will all have
different solubilities in the diluent
• The analyte-Its reactivity with diluent
• Source of impurities-synthetic or diluent-in situ
14. METABOLIC AND DEGRADATION PATHWAYS
In silico
• META SITE
• METABOL-
EXPERT
• METEOR
• METABOLISM
• ACD/MS
DATABASES
• MDL
METABOLITE
• DRUG BANK
• GEN BANK
MET ID-LCMS
• METABOLYNX
• MET ID
• METWORKS
• METABOLITE
PREDICT
In vitro
MODELS
• RAT
• MOUSE
• MONKEY
• LIVER
FRACTIONS
• CYP 450
ISOZYMES
15. DETECTOR CONSIDERATIONS
• The UV spectra of target
analyte and impurities must be
taken and overlaid with each
other.
• A wavelength must be chosen
such that adequate response is
obtained for the active and
that at least a 0.05 v/v%
solution of the active at target
concentration could be
quantified (S/N greater than
10)
UV
PDA
MASS
NMR
ELSD
FLUORESCENCE
ELECTROCHEMICAL
LIGHT SCATTERING
REFRACTIVE INDEX
FLAME IONIZATION
CORONA CHARGED AEROSOL DETECTION
16.
17. • The spectral homogeneity of the peak of interest must be taken into
consideration.
• Diode array spectra at least three points across the peak should be
taken to ensure the peak is spectrally homogenous
18. SOLUTION STABILITY AND SAMPLE PREPARATION
• Stability of solution in the diluent is important
• Sample processing should be done carefully at desired temperatures
• Sample preparation is a critical step of method development
• Centrifugation effects (speed & time)
• Filtration effects
19. MOBILE PHASE CONSIDERATIONS
• Alteration of the mobile-phase pH is one of the greatest tools in the
“chromatographers toolbox” allowing simultaneous change in
retention and selectivity between critical pair of components.
• Purity of the solvents systems
• Instrument suitability
• Economic considerations
24. MOBILE PHASE pH
BASED ON ANALYTE
IONIZABLE
ONE IONIZABLE
CENTER
pH = ±1 pKa OF THE
ANALYTE
MORE THAN ONE
IONOZABLE CENTER
Log P OF THE
DRUG
NEUTRAL
BASED ON
RUN TIME
FAST
ANALYSIS
IN IONIC STATE
IF MATRIX IS
COMPATIBLE
IF MATRIX IN IN-
COMPATIBLE
MOBILE PHASE
ADDITIVES(<5%)
ACIDIC
MODIFIERS
BASIC
MODIFIERS
ION-PAIR REAGENTS
pH-ADJUSTMENT
CONVERTED TO
NEUTRAL FORM
SLOW
ANALYSIS
IN NEUTRAL STATE
25.
26. STRENGTHS WEAKNESSES
OPPORTUNITIES THREATS
pH = pKa – 2
the acid is 1 % dissociated
(mostly neutral)
pH = pKa
the acid is 50 % dissociated
(equal amounts of ionized and
neutral)
pH = pKa + 2
the acid is 99 % dissociated
(mostly ionized)
pH = pKa – 2
the base is 99 %
dissociated
(mostly ionized)
pH = pKa
the base is 50 % dissociated
(equal amounts of ionized
and neutral)
pH = pKa + 2
the base is 1 % dissociated
(mostly neutral)
27. CHOICE OF BUFFERS
• In order to develop rugged HPLC methods, knowledge of choosing the
right buffer is very important.
• Buffers that are selected should have a good buffering capacity at the
specified mobile-phase pH.
• The concentration of the buffer should be at least 10mM.
• Optimum buffering capacity occurs at a pH =pKa of the buffer.
• In general, most buffers provide adequate buffering capacity for
controlling mobile-phase pH only within ±1 unit of their respective
pKa.
• Also, buffers are great media for growing bacteria. It is recommended
to have at least 10 v/v% of organic in the aqueous phase to prevent
bacterial growth.
28. GENERAL CONSIDERATIONS FOR BUFFER
• The type of buffer that is chosen will depend on the wavelength of the method
and the concentration of organic in the mobile phase.
• A judicious choice of type and concentration of buffer must be made to ensure
mobile-phase compatibility.
• Purity of buffer should be taken into consideration.
• Phosphate is more soluble in methanol/water than in acetonitrile/water or
THF/water.
• Some salt buffers are hygroscopic. It leads to improper final concentrations.
• Ammonium salts are generally more soluble in organic/water mobile phases
than potassium salts, and potassium salts are more soluble than sodium salts.
29. • TFA can degrade with time, is volatile, absorbs at low UV wavelengths, and is not
a buffer at pH > 1.5.
• Citrate buffers can attack stainless steel.
• At pH greater than 7, phosphate buffers accelerates the dissolution of silica
and severely shortens the lifetime of silica-based HPLC columns. If possible,
organic buffers should not be used at pH greater than 7.
• Ammonium bicarbonate buffers usually are prone to pH changes and are usually
stable for only 24 to 48 hours.
• After buffers are prepared, they should be filtered through a 0.2-µm filter.
• Mobile phases should be degassed if an on-line degasser is not available on
the HPLC system.
31. CONCENTRATION OF BUFFERS
• A buffer concentration in the range of 10 to 50 mM is adequate for most
reversed-phase applications.
• However, sometimes the concentration of the buffer does lead to improvement
of peak shape.
• The concentration should also be low enough to avoid problems with
precipitation when significant amounts of organic modifiers are used in the
mobile phase.
• It is seldom advisable to use a buffer concentration more than 100 mM and less
than 10 mM.
• It is mainly ionic strength and not a buffer capacity that is governing the peak
distortion in less than 10 mM concentration range—at these conditions the
solvation equilibrium is slow, causing peak distortion.
32. PRACTICAL ASPECTS OF PREPARING A
BUFFERED MOBILE PHASE
• Define the appropriate pH for the separation and then select an
appropriate buffer.
• Prepare an aqueous buffer solution of the desired concentration and
pH.
• Measure the pH of the solution and adjust, if necessary, to the
desired pH with dilute acid or dilute basic solution.
• If you are performing an isocratic separation, combine the aqueous
buffer solution with the appropriate organic modifier (e.g., methanol
or acetonitrile) to produce the desired mobile phase and let the
solution come to equilibrium.
33. CHOICE OF ORGANIC MODIFIER
• Selection of the organic modifier type could be viewed as relatively simple
• The usual choice is between acetonitrile and methanol (rarely THF)
• In short, methanol shows more predictable influence on the analyte elution
• The viscosity of water/organic mixtures should be considered as an
additional parameter in the selection of organic modifier.
• The stability of the mobile phase should also be considered. THF is known to
form peroxides.
35. SEPARATION MODES
• Isocratic Versus Gradient Separations
• Traditionally, isocratic separations are deemed as more reproducible
than gradient separations.
ISOCRATIC
CONSTATNT EQUILIBRIUM
CONDITIONS
PREDICTABLE SEPARATIONS
LESS NUMBER OF SAMPLES
LOW PEAK CAPACITY, BROAD
PEAKS
GRADIENT
HIGH SEPARATION POWER
LESS PEAK WIDTH
TAILING DEPENDS ON THE
STRONGER ELUENT
FAST RATES OF SEPARATION
36. CHOICE OF STATIONARY PHASE
Knowledge of the Sample
• Structure of sample components?
• Number of compounds present?
• Sample matrix?
• pKa values of sample components?
• Concentration range?
• Molecular weight range?
• Solubility?
• Other pertinent data?
Column Chemistry
(bonded phase, bonding type,
end capping, carbon load)
37. Goals for the Separation
• Max. resolution of all components?
• Partial resolution?
• Fast analysis?
• Economy (low solvent usage)?
• Column stability and lifetime?
• Preparative method?
• High sensitivity?
• Other goals?
Column Physics
(particle bed dimensions,
particle shape, particle
size, surface area, pore
size)
40. COLUMN SELECTION GUIDE
ANALYTESTRUCTURE
LOG P
STATIONARYPHASE
HYDROPHOBIC-
+VE LOG P-RP
COLUMN-C8 OR
C18
POLAR
ANALYTES-NP
COLUMN
CHIRAL-SUITABLE
CHIRAL COLUMN
SORBENTSPECIFICATIONS
MATRIX
TOLERABILITY
MS SUITABILITY
PARTICLE SIZE
pH-STABILTY
COLUMDIMENSION
LENGTH X i.d
GUARD
PREPARATIVE
SEMI-
PREPARATIVE
PRE-COLUMN
METHOD
DEVELOPMENT
USPCLASSIFICATION
L1-L60
41.
42. CHOOSING RIGHT COLUMN FORMAT
PARTICLE SIZE
• SMALL-HIGH
SEPARATION
EFFICIENCY & HIGH
RESOLUTION
• LARGE-OFFER FAST
FLOW RATES,LESS
PRONE TO
CLOGGING
• 5μm IS BEST OPTION
PORE SIZE
• PORE-LARGE
ENOUGH TO
COMPLETELY
ENCLOSE TARGET
• SMALL-HIGH
SURFACE
AREA,HIGHER
CAPACITY
• LARGE-SMALL
SURFACE
AREA,FASTER
EQUILIBRATION,GRA
DIENT MODE,
PORTEINS
CARBON LOAD
• AMOUNT OF
FUNCTIONAL
BONDED PHASE
ATTACHED TO THE
BASE MATERIAL
• LOW-WEAKLY
HYDROPHOBIC,LOW
RT
• HIGH-GREATER
RESOLUTION,HIGHER
CAPACITIES,HYDROP
HOBIC COMP.
END CAPPING
• END CAPPING THE
BONDED PHASES
MINIMIZES
SECONDARY
INTERACTION WITH
FREE SILANOL
GROUPS
• USE IF
INTERACTIONS WITH
POLAR COMPOUNDS
IS NOT REQUIRED
• NON-END CAPPING-
POLAR
SELECTIVITY,STRONG
RETENTION OF
POLAR COMP.
43. Feature Utility
5-µm totally porous particles Most separations
3-µm totally porous particles Fast separations
1.5-µm pellicular particles
Very fast separations (especially
macromolecules)
± 50% (from mean) particle size
distribution
Stable,reproducible,more efficient with
low column pressure drop
7-12nm pores, 150-400 m2/g (narrow
pore)
Small molecular separations
15-100nm pores, 10-50 m2/g (wide
pore)
Macromolecular separations
COLUMN SELECTION GUIDES:
• Chromatographic Columns Online Database-USP NF
• WATERS
• AGILENT
• PHENOMENEX
• THERMOSCIENTIFIC
44. EQUILIBRATION & OTHER FACTORS
• Equilibration with mobile phase is very important in gradient elution
• The time needed to equilibrate the column is determined by flow rate
• Lower the flow rate, longer the equilibration time
• Flow rate has to be selected based on separation of impurities, column back pressures
and retention times.
• Generally flow rate shall not be more than 3.0ml/min. High flow rate reduces analysis
time
• Select the flow rate which gives least RT & back pressure, good peak symmetry &
separation of impurities.
• In most cases ambient temperature is used to optimize the chromatographic conditions.
• If peak is asymmetric with different mobile phase and column combinations then
temperatures above ambient can be used.
• Increase in temperature decreases analysis time
• Increasing flow rate, increasing temperature (up to column stability limit at a particular
pH), increasing the concentration of the organic eluent, and using shorter columns with
narrower dimensions may be used to obtain more desirable run times.
45. METHOD OPTIMIZATION
OPTIMIZATION
CONSIDERATIONS
• MOBILE PHASE pH
• ORGANIC SOLVENT COMPOSITION
• FLOW RATE
• GRADIENT ELUTION
• TYPE OF COLUMN
• COLUMN TEMPEARTURE
DATA SYSTEMS
• DRYLAB
• AMDS-WATERS
• CHROMSWORD
• AUTOCHROM