HPLC:

Volatility and Thermal Stability are the two main reasons due to which
HPLC is used instead of GC for analysis.
Efficient require less time for separation.
Column is densely packed with finely divided stationary phase (3-10
microns); hence mobile phase requires to flow at high pressure (300psi)
via column.

Theory:
Separation depends upon-
Nature of sample
Nature of stationary phase
Nature of mobile phase
Retention time: time taken by sample component to travel from point of
injection to end of column, unique to analyte.
Component with high affinity for mobile phase move fast than component with
more affinity for stationary phase (move slowly, more retention time, retained
on column for longer time).
Gaussian Curve & Chromatographic Peak:
Upon elution solute not elute at a singular time, rather elute over time.

Otherwise gaussian curve won’t be observed.

 Some molecule travel fast (starting part), come out first while most
travel with average speed (middle region) and rest come at last (end
part).
When compressed it looks like-

Peak Broadening:
As solute travel via column peak broadening occurs, causing poor
resolution
For optimal separation symmetrical chromatographic peak must be
obtained, so need to limit broadening.
Plate Theory:
By Martin & Synge in 1941
Says that column consist of series of discrete horizontal layers
(theoretical plates)
Solute equilibrate at each plate b/w stat and mob
Solute migrate stepwise b/w one plate to below plate
Efficiency of separation column increase as no. of plate increase and H
decrease.
N=L/H
H= Height of equivalent plate
N= no. of theoretical plates
L= length of column
Imaginary plates only
Rate Theory:
By van Deemter in 1956.
Describe shape and width of peak
Based on random walk mechanism for migration (STOP/GO) of solute via
column
Explain effect of mob velocity and adsorption capacities of stat, they
affect width of band (peak)
Shows relation b/w mob velocity and adsorption capacities of stat with
retention time.
STOP- adsorbed on stat
Go- present in mob
Rapidly exchange adsorbed and desorbed state
Migration of one solute is irregular and independent of other molecules
Band width increase upon migration of solute down the column
Band width is directly prop to retention time and inverse to mob velocity
H= A+ B/µ + Cµ
A- eddy diffusion B- Longitudinal Diffusion C- Mass Transfer µ- linear
velocity of mob
Eddy Diffusion: multiple path effect due to irregularities in flow velocity
and path around packing particles. (A= λdp) λ is function of packing
uniformity and column geometry. If irregular packing then unequal path
length occur. Green color path largest while blue smallest path. To
minimize it mean diameter of particle in packing should be as small as
possible and be uniformly packed.

Longitudinal Diffusion: axial diffusion, solute tend to diffuse to all

direction (from high to low conc). B= 2 ƳDM , Ƴ is obstruction factor &
 depend upon column packing & DM is solute diffusion coefficient in mob.
So broad band occurs if mob velocity too low, DM would be high, solute
disperse axially and elute slowly. Minimized by optimizing mob velocity.
Mass Transfer: Cs is resistant to mass transfer at solute to stat interface,
directly proportional to thickness of stat (df) and inverse to diffusion
coefficient hence if stat particle is small less resistant and elute fast. But
if mob velocity high and resistant is more i.e. large particle of stat so
solute spend more time in stat causing broad band. Cm is resistant to
mass transfer b/w adjacent stream line of mob and directly proportional
to square of particle diameter(packing) and inverse to DM of solute in
mob.
Average Linear velocity: if low then B would be more, if high then Cs
would be more. Both cause broad band hence velocity should be
optimal.

Modes of Separation in HPLC:

1. Normal Phase:
separation based on polarity.
solute interacts with stat.
stat. is polar e.g. silica, alumina, bonded PVA.
mob. Is non polar e.g. heptane, hexane
so non-polar solute moves faster, elute fast
polar solute elutes slow
for separation of oil soluble vit., essential oils.
Not much use in pharma, since most drugs are polar.
If polar substance used, retention time would be more, broader
peak.
2. Reverse Phase
Stat. is non polar e.g. hydrophobic bonded octadecyl C-18, octyl C-
8
Mob. Is polar e.g. methanol, acetonitrile
Polar solute elutes fast, non-polar elute slow.
Separation occurs as mob. Enforce solute into hydrocarbanous
bonded layer of stat.
3. Adsorption:
Stat. is solid, competitive interaction with solute.
 4. Liquid-Liquid:
Mob. Is liquid coated on porous support material, this liquid is
insoluble in stat.
Analytes partition b/w liquid stat and mob.
5. Ion-exchange:
Cation or anion exchanger resin as stat.
Mob. is electrolyte solution
6. Ion-pair
7. Size Exclusion
8. Affinity

Instrumentation of HPLC:
1. Solvent Reservoir:
High quality borosilicate
0.5 to 1 L
Nylon filter is used to filter solvent then filled in reservoir
Teflon tube carries solvent to pump and from detector to waste.
2. Solvent Degasser:
by vacuum remove soluble gas.
Enter mixing chamber get uniformly mixed
3. Solvent Delivery System:
Known as pump, imp part of HPLC
Apply pressure on solvent to pass via dense column
Capable of output of 5000psi and no pulses in flow rate
Composed of chemically resistant material to solvent
Flow rate required 0.5-2ml/min.
Stainless steel tube carries solvent further to detector
 Constant Pressure Pump: pneumatic pump, pressure from a gas
cylinder delivered to piston, it drives mob. Pressure prop to ratio
of area of 2 piston and pulse free flow.

Constant Displacement Pump: syringe type, have large syringe

like chamber with a plunger. Motor drive the plunger forward and
backward. Good flow rate independent of viscosity and back
pressure, but limited capacity 250ml.
Reciprocating Piston Pump: motor driven piston immersed in
hydraulic chamber (contain oil), piston move to and fro.
Economical and most popular.

4. Sample injector:
Manual or automatic
 Rheodyne Injector: micro-volume sampling valve, most popular,
has fixed volume of sample loop 5 to 20 µL. Sample injected via
hypodermic needle.

5. Columns:
Guard Column: prolong life of analytical column, inserted ahead of
them. Relatively short up to 5cm have stat. like analytical. Protect
analytical from particulate contamination of mob. Heart of HPLC,
but cause broad band as increase column length so in line filter is
alternate to guard column.
Column have heavy wall, glass lined metal tubing or stainless-steel
tubing due to pressure and chemical action of mob.
End of column have stainless-steel frits to retain packing.
Usually std. column is 10-3- cm long.
Column Packing:
▪ Microporous Support: particle size 5–10-micron, adsorbent
like silica, alumina, resins used.
▪ Pellicular Support: porous particles coated on inert solid
core (glass bead) of 40-micron diameter.
 ▪ Bonded Phases: chemically bonded stat on inert support,
coated stat may get washed off with mob hence bonded
phase developed.

Types of columns:
▪ Standard Column: 10-30cm long with internal dm of 4-5mm.
3–5-micron particle is packed. NP-HPLC packed wit silica gel.
RP-HPLC packed with octadecyl silanol C18, octyl silanol C8
or butyl silanol C4. Good efficiency, capacity for both
analytical and preparative work.
▪ Radial Compressed Column: flexible wall, dm of 8mm,
cartridge is held in plastic tube require less pressure and
short time of analysis and economical.
▪ Narrow Bore Column: 1 or 2mm internal dm hence
increased signal, few of these columns can be connected to
get a long column (higher plate count) since smaller dm can’t
do with others.
▪ Short fast Column: 3-6cm long with internal dm of 4-5mm,
QC work fast speed with high sensitivity.
6. Detectors:
 Detector Type Principle Advantages Disadvantages Typical
Applications
UV-Vis Detector Measures High sensitivity, Limited to Pharmaceuticals,
absorbance of universal for many chromophoric food analysis
UV/visible light compounds compounds
Fluorescence Measures emitted Highly sensitive, Requires Trace analysis,
Detector light from low detection fluorescence; environmental
fluorescent limits not universal monitoring
compounds
Refractive Measures changes Universal; can Lower Sugar, lipids
Index Detector in refractive index detect non- sensitivity; analysis
chromophoric slower
compounds response time
Conductivity Measures ionic Good for ionic Not suitable for Ion analysis,
Detector conductivity of species non-ionic pharmaceutical
eluent compounds compounds
Mass Measures mass- High specificity, Expensive, Proteomics,
Spectrometer to-charge ratio of structural complex, metabolomics
ions information requires
ionization
Electrochemical Measures current Highly sensitive Limited to Environmental
Detector produced by redox for electroactive specific types of analysis, food
reactions species compounds safety

Light Scattering Measures Useful for large Requires Polymers,

Detector scattered light molecules/colloids calibration, not proteins
from particles widely used