c

c 

In gas chromatography, only about 20% of known compounds

were lend themselves to analyse either because they are insufficiently
volatile and cannot pass through the column or because they are
thermally unstable and decomposes under the conditions of separation
and one of the early problems with liquid chromatography was the slow
rate at which the analysis took place. Early methods used gravity feed,
and it was not uncommon for an analysis to take several days to
complete. This led to great delay, but also the excessive time on the
column inevitably led to loss of resolution by diffusion, and so on.
Consequently, for a number of years liquid chromatography was not
widely used as a mean of separating organic compounds. These
problems were largely overcome by the advent of HIGH-
PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) because it is
not limited by sample volatility or thermal stability. High -performance
liquid chromatography (HPLC) is a form of liquid chromatography to
separate compounds that are dissolved in solution. HPLC instruments
consist of a reservoir of mobile phase, a pump, an injector, a separation
column, and a detector. Compounds are separated by injecting a plug
of the sample mixture onto the column and here in this system pressure
is applied to the column, forcing the mobile phase through at much
higher rate. The pressure is applied by using a pumping system. The
action of the pump is critical, since it must not pulsate and mix up the
sample being separated in the solvent, causing it to lose resolution.
Development of pumps has proceeded quite quickly over the last
several years, and now it is possible to achieve good resolution under
the conditions required for HPLC.
HPLC is able to separate macromolecules and ionic species, labile
natural products, polymeric materials, and a wide variety of other high-
molecular-weight polyfunctional groups. With an interactive liquid
mobile phase, a parameter is available for selectivity in addition to an
active stationary phase. Chromatographic separation in HPLC is the
result of specific interactions between sample molecule with both t he
stationary and mobile phases. These interactions are essentially absent
in the mobile phase of gas chromatography. HPLC offers a greater
variety of mobile phases, which allow a greater variety of these
interactions and more possibilities for separation. Sample recovery is
easy in HPLC. Separated fractions are easily collected by placing an
open vessel at the end of the column. Recovery is usually is
quantitative (barring irreversible absorption on a column), and
separated sample components are readily isolated from the mobile
phase solvent. In addition to usual type of organic compounds, liquid
column chromatography can handle separations of ionic compounds,
labile naturally occurring products, polymeric materials, and high
molecular-weight polyfunctional compounds
All the forms of liquid chromatography are migration processes
where sample components are selectively retained by a stationary
phase.
High Performance Liquid Chromatography (HPLC) was developed
in the late 1960s and early 1970s. Today it is widely applied for the
separations and purifications in a variety of areas including
pharmaceuticals, biotechnology, environmental, polymer and food
industries.
HPLC has over the past decade become the method of choice for
the analysis of a wide variety of compounds. Inn HPLC the separation
of a mixture into its components depends on different degrees of
retention of each component in the column.? The extent to which a
component is retained in the column is determined by its partitioning
between the liquid mobile phase and the stationary phase. In HPLC this
partitioning is affected by the relative solute/stationary phase and
solute/mobile phase interactions. Thus, unlike in gas chromatography,
the ability to manipulate the character of both the stationary phase and
the mobile phase decreases the need for a large number of stationary
phases in liquid chromatography. Knowledge of the molecular structure
of the sample components can be very helpful in the selection of a
liquid chromatographic method.
In general, HPLC is used for the following purposes-
‡ Separation of organic, inorganic, biological compounds,
and thermally labile compounds.
‡ Qualitative and quantitative analysis of the compounds.
HPL 


HighPressureLiquidhromatography;
HighPricedLiquidhromatography;
Hewlett PackardLiquidhromatography;
HighPerformanceLiquidhromatography;
HocusPocusLiquidhromatography;
HighPatienceLiquidhromatography;
High Performance Liquid Chromatography (HPLC) is one mode of
chromatography the most widely used analytical technique.HPLC
utilizes a liquid mobile phase to separate the components of a mixture.
These components (or analytes) are first dissolved in a solvent, and
then forced to flow through a chromatographic column under a high
pressure. In the column, the mixture is resolved into its components.
The interaction of the solute with mobile and stationary phases can be
manipulated through different choices of both solvents and stationary
phases. As a result, HPLC acquires a high degree of versatility not
found in other chromatographic systems and it has the ability to easily
separate a wide variety of chemical mixtures.


Pc cPLE
HPL 
Different types ofHPLC act on the basis of two laws:-
Ɣ Adsorption law
Ɣ Partition law
hen a solid surface is exposed to a gas or a liquid, molecules
from the gas or the solutionphase accumulated or concentrated
at the surface of the solid.´ this phenomenon is called as
adsorption.
The Partition Law states that ±
he ratio of the partition or
distribution of a compound between two immiscible phases
remain constant.´The constant is called as partition constant or
distributionconstant.´
For a compound distributing itself between equal volumes of two
immiscible solvents A and B, the value for this coefficient is a constant
at a given temperature and is given by the expression:-

†






 d=
†







The distribution of a compound can be described not only in of its

distribution between two solvents, but also by its distribution between
any two phases, such as solid/liquid or gas/liquid phases.
 It i t tt l i f t i l
i it l l t t f t ti l l t 
it l t , lt t li it t t l t f l t
l f i . A t f t ti l l t i
l t t t f f t t ti it f ll t t t
ll t ti l i f t t ti , tt t l ti .
f t t l , t ll i , t t t i t t l t
fl . All f t f f t l t l it
l i t f t l f l tt t l
ll l f li i t .

t f t i i t fl t i
l ti l l t i i t ti f i l i
iff i . f f t fl t i t i l
it t l i i ffi i t t
t ti t t f t t ti , t t ll
i l t fl i ii l ti . I t t t
ti l t i l t i t l ,
i lt i t il ilit f ti l i t ti
i it t t f i t
i i li l fl t . l t , i
i ti , titi , i , i l i
ffi it t , lt i f t tt l ti
l i P t t l , f l
til f ft t .
 P ll f
3 t t , i
t t f l . i i t i t ti
li fl t f t il . It i t t t i i
t ll t t ti l l t i t. It i i i t ll i
t t l ti i /V i l .

PES P C:-
t l if li i l t . If
t i l ifi ti i t t f t i t t f
ti
1. Cl i l P C l ifi t t f t
t ti t ti ,
ifi , i ²
A ti t 
li i li i t 
I t 
Si t i l i t 
Affi it t 
B t 
I ii t 
.  P C l l ifi t t f t
t ti t ti ,t i ²
 Chiral HPLC; 
ƔCapillary HPLC;
All above mentioned chromatographic techniques are briefly described
below:-

SP
c  H
 PH  Lcc

LccH
 PH ?
????In adsorption chromatographythe stationary phase is an adsorbent
(like silica gel or any other silica based packings) and the separation is
based on repeated adsorption-desorption?steps. 
If the sample is water insoluble, possesses an aliphatic or aromatic
character, and has a molecular weight less than 2000 Daltons for all of
the sample components adsorption chromatography and liquid liquid
chromatography are possibilities. Adsorption chromatography works
best for class separation or for the separation of isomeric compounds.
The technique of liquid liquid chromatography works better for the
separation of homologous compounds. Functional groups that are
capable of strong hydrogen binding are retained strongly in adsorption
chromatography; however liquid-liquid chromatography provides an
alternate method for the separation of such compounds. These will be
samples that posses medium polarity and are soluble in weakly polar to
polar organic solvents. In general the separation of non electrolytes in
liquid-liquid chromatography is achieved by matching the polarities of
the sample and stationary phase and using a mobile phase that has
markedly different polarity.
Concerning this type of chromatography, two modes are defined
depending on the relative polarity of the two phases:-
Ɣ Normal-phase chromatography;
Ɣ Reversed-phase chromatography;
In normal phase chromatography, the stationary bed is
strongly polar in nature (e.g.; silica gel), and the mobile phase is
nonpolar (such as n-hexane or tetrahydrofuran). Polar samples are
thus retained on the polar surface of the column packing longer than
less polar materials.
In reversed phase chromatography, is the inverse of this.
The stationary bed is nonpolar (hydrophobic) in nature, while the
mobile phase is a polar liquid, such as mixtures of water and methanol
or acetonitrile. Here the more nonpolar the material is, the longer it will
be retained.

ý E PHASEHA
APH 
The most widely used column packings for liquid-liquid partition
chromatography are those with chemically bonded, organic stationary
phases. They replace the classical packings in which the stationary
liquid phase coated with a supporting material. Partition occurs
between the bonded phase and a mobile liquid phase.
Bonded ±phase supports are made from silica by the covalent
attachment of an organic hydrocarbon moiety to the surface. Supports
include large porous silica gel, porous layer beads, and micro particles.
Bonded-phase packings are quite stable because the stationary phase
is chemically bound to the support and cannot be easily removed
during use.

Si OH + ClSi (CH) R Si O Si(CH) R +HCl

The siloxane (Si O Si C) type of bond has become the

standard for commercial bonded phases. It is formed by the
reactions of di-(or tri-) alkoxysilane with the surface of silanol
groups of fully hydroxylated silica gel, as either pellicular or
totally porous supports.

c EXHA EHA
APH 

In ion-exchange chromatography the stationary bed has ionically
charge- bearing functional groups attached to a polymer matrix. The
functional groups are permanently bonded ionic groups associated with
counterions of the opposite charge. The most common retention
mechanism is the simple exchange of sample ions and mobile-phase
ions with the charged group of the stationary phase. This technique is
used almost exclusively with ionic  or ionisable samples, when the
sample is water soluble and molecular weights are less than 2000
Daltons. The stronger the charge on the sample, the stronger it will be
attracted to the ionic surface and thus, the longer it will take to elute.
Some ion-exchange packings be negatively charged groups and
are used for exchanging cationic species and positively charged
packings are used for exchanging anionic species. The most commonly
used functional groups are the sulphonate type exchangers for cation
exchange & the quaternary amine type exchanger for anion exchange.
O
O H
S
O

R
R O

S O

OH
R

HO S R
O
O

Sulfonicacidsexchangeresinforcationexchange.
 
H

H   -
R l
 
H

H  
H  
l
-

 
H  
H  H

 -  
l H

Strong base uaternary amine) exchange resin for anion

exchange.
There are no any effect of pH of mobile phase on the exchange
properties because they are strong acids or bases in the -H or ±OH
form respectively. These exchangers have considerable affinity for
heavy metal cations and, to a lesser extent, for alkaline earth cations.
O H


H   O

O
O H

Aminodiacetate
Aminoacetate exchanger fined its use as a column packing for ligand
exchange.

c PAcc HA
APH 
Ion- pair chromatography, is considered as a subset of reverse-
phase chromatography, can deal with ionized or ionisable species on
reverse-phase columns. The method overcomes difficulty in handling
certain samples by the other liquid chromatography methods; these are
samples that are very polar, multiply ionized, and/or strongly basic. In
ordinary reverse-phase HPLC, organic ions show poor peak shapes
and inadequate retention. The ion suppression method is also limited to
the pH range 2.0-7.5 by the instability of stationary bonded phases
outside this pH range. Ion-exchange packings offer limited choices with
little ability to vary selectivity by changing the column packing.
In ion-pair chromatography an ion-pair reagent (a large organic
counterion) is added low concentration (usually 0.005M) to the mobile
phase. The ion-pair reagent is itself ionized. One ion of the reagent is
retained by the stationary phase, thus providing the otherwise neutral
stationary phase with its charge. This charged stationary phase can
then retain and separate organic solute ions of the opposite charge by
forming a reversible ion-pair complex (a columbic association species
formed between two ions of opposite electrical charge) with the ionized
sample as represented by the following equilibrium:

RCOO + R4N!  [ R4N!, OOCR] "ion pair

-
Here it is assumed that the solute ion is a caboxylate anion, RCOO
and that the counter ion is a quaternary ammonium ion, R 4N+. Thus
with a suitable counterion, ionic or nonionizable compounds can be
converted to electrically neutral compounds that will partition between
the mobile and nonpolar stationary phases. At the, same time, the
stationary phase will not have lost any of its ability to retain and
separate nonionized organic substances.
There are two fundamental models have been proposed for the
mechanism of the ion-pair chromatography-
2 The first postulate is that the solute molecule forms an ion pair
with the counterion in the mobile phase. This uncharged ion pair
then partitions into the lipophilic stationary phase(C-18).
  
 


i

i
 
 




ilica particles N+

2 The second is that the counterion partitions into the stationary

phase, or is ³loaded´ onto the bonded reverse-phase packing,
with its ionic group oriented at the surface. This produces two
possibilities for the material to be chromatographed. It can be
attracted to the hydrocarbon partition in the usual revese-phase
manner, or it can interact in an ion-exchange mode. It is quite
likely that the true mechanism involves both postulates but is
futher complicated by adsorption and micelle formation. Whatever
the mechanism, ion-pair chromatography allows for unique
separations not otherwise obtainable by either reverse phase or
ion-exchange.

 
 
i  
i

  + -


+ 
N 

N
N

N N
 ?
Silica particles
ounterionalkylsulfonate)loadedontostationaryphase.

ig:-Mode of selection of type of liquid chromatography for sample
molecules having molecular weight less than 2000.

Sc#E EXLSc   EL PEEA
c 

HA
APH
In size exclusion chromatography  the column is filled with
material having precisely controlled pore sizes, and the sample is
simply screened or filtered according to its solvated molecular size. It is
especially applied when it is known or suspected that the molecular
weight exceeds approximately 2000 daltons for some or all of the
sample components. Larger molecules are rapidly washed through the
column; smaller molecules penetrate inside the porous of the packing
particles and elute later. Mainly for historical reasons, this technique is
also called gel filtration or gel permeation chromatography although,
today, the stationary phase is not restricted to a "gel".
Much work in the food and beverage industry, or physiological
samples in which most of the Krebs cycle acids (tricarboxylic acid
cycle) are present, is easily done with an anion exclusion column.
Wine, beer, fruit juices, and many dairy products are quickly analyzed
with minimal sample preparation (usually only filtration or
centrifugation). It also used in separation of oligosaccharides and sugar
alcohols using an anion exclusion column in the calcium ionic form with
water as eluent at temperature of 900C. Maltotriose and higher
oligosaccharides are separated from mono- and disaccharides by steric
exclusion effects.
 c c C  P:-
 l f ffi it t i l t
l t tt t f i ili i i ffi it l ll
li t li t. l i t t
l , l t l t t t l ti l i t t l t
li i t i ;t t l t l t it t t ti .
ti l it t l i i t t i l t i
i l i l t . t i l t l t f t
l i t il iti . j t
f t i t i i it t ifi it , i it i
i l ti i ti it i l i i l t .

l ti i l
ifi f f t l i , ll l , t ,
l l
i , i ti f t l , i
l . ti l ffi i tl ili t i t
ifi i i f l t t l t t t ti l
i l t . A ti it i f li
tit ti i i l t l l l .
P ti t t il l i ll
i l

 
B 

= l l l
 Agarose is a very popular matrix; its porous meshwork can be
strengthened by cross-linking it with 1-chloro-2, -epoxypropane.
The affinity ligand can be antibodies, enzyme inhibitors, or other
molecules that reversibly and bioselectively bind to the complementary
analyte molecules in the sample. The affinity ligands are of two types;
one is Ligand specific, and other is Group-specific. Group-specific
ligands are used for the separation of ligands such as, proteins,
including


E
E
c EHA cS
In general, HPLC is a dynamic adsorption process. Analyte
molecules, while moving through the porous packing bead, tend to
interact with the surface adsorption sites. Depending on the HPLC
mode, the different types of the adsorption forces may be included in
the retention process:
Ɣ Hydrophobic (non-specific) interactions are the main ones in
reversed-phase separations.
Ɣ Dipole-dipole (polar) interactions are dominated in normal phase
mode.
Ɣ Ionic interactions are responsible for the retention in ion-exchange
chromatography.
 All these interactions are competitive. Analyte molecules are
compete with the eluent molecules for the adsorption sites. So, the
stronger analyte molecules interact with the surface and the weaker the
eluent interaction, the longer analyte will be retained on the surface.
SE size exclusion chromatography) is a special case. It is the
separation of the mixture by the molecular size of its components. In
this mode any positive surface interactions should be avoided (eluent
molecules should have much stronger interaction with the surface than
analyte molecules). Basic principle of SEC separation is that the bigger
the molecule, the less possibility for her to penetrate into the adsorbent
pore space, so, the bigger the molecule the less it will be retained.


HcALHA
APH
In chiral chromatography the following terms are used-
hiral stationary phase  A stationary phase which incorporates a
chiral selector. The term chiral stationary phase does not necessarily
mean that the stationary phase itself is chiral (although in practice it
usually is) but that the stationary phase is used to separate chiral
substances. Two substances can only be separated if their standard
energy of distribution differ, which means that their standard enthalpies
and/or their standard entropies of distribution also differ. In general, the
standard enthalpy reflects the net difference in the interactive forces on
the molecule in the two phases (polar, dispersive and ionic interactive
forces) whereas the standard entropy reflects their spatial disposition
and, thus, their probability and proximity of interaction. Thus, for any
chiral separation the stationary phase is chosen such that the spatial
arrangement of its composite atoms results in the probability or
proximity of interaction differing significantly between the two
enantiomers to be separated. If not a constituent of the stationary
phase as a whole, the chiral selector can be chemically bonded to
(chiral bonded stationary phase)or immobilized onto the surface of a
solid support or column wall (chiral coated stationary phase),or simply
dissolved in the liquid stationary phase.
hiral selector The chiral component of the separation system
capable of interacting enantioselectively with the enantiomers to be
separated.
hiral additive The chiral selector which has been added as a
component of a mobile phase or electrophoretic medium.
hiralmobilephase A mobile phase containing a chiral selector.
Chiral chromatography is highly dependent on the column, which
has seen many recent improvements, and the detector.The chiral
chromatography is specially used for the separation of enantiomeric, or
diasteriomeric compounds (drugs). It is a relatively new technique of
HPLC.

APcLLAHPL
Capillary HPLC uses smaller column internal diameters than
conventional HPLC. Smaller ID columns, for fixed amounts of injected
material, produce taller peaks. Taller peaks provide better detection
limits for mass spectrometry and other concentration sensitive
detectors. For the same amount of material injected, the peak height is
inversely proportional to the cross sectional area of the column. The
use of smaller ID columns requires careful planning if you are used to
normal 4.6 mm columns. Liquid chromatography/mass spectrometry,
LC/MS, is a revolutionary tool in the chemical and life sciences. LC/MS
is accelerating chemical research by providing a robust separations
and identification tool for chemists and biologists in diverse fields.
LC/MS is best done with capillary HPLC. This General Introduction to
capillary HPLC is designed to provide a practical survey of the day-to-
day issues in solving chemical problems using HPLC and to give you
the necessary information to use the Agilent 1100 Capillary HPLC and
Ion Trap SL.

PES
EL
c S 
There are two elution types-
2 Isocratic elution.
2 Gradient elution.
In the first type constant eluent composition is pumped through the
column during the whole analysis. This is cSA
cEL
c .
In the second type, eluent composition (and strength) is steadily
changed during the run. This is AcE
EL
c .


A
S c
LE c 
HE E
E
c 
cE 
SEPAA
c c
HLccHA
APH 
There are several factors influencing the separation through HPLC are
given below:
2 Column length,
2 Sample distribution between stationary and liquid phases,
2 The selection of the stationary and liquid phases,
2 Temperature of the column,
2 Pressure drop of the column,
2 Viscosity of solvent,
2 Particle diameter of stationary phase,
EFFECT F TEMPERTURE F CO UMN ON TE
RETENTcONTcME:-
I iti l P l , i ti i t t
i ifi t i t ti ti , i lit ti l i
iffi lt ff ti t i i f tit ti t .
El t t t t il
it , i i t f , i l
l ilit lt i it tt l ti f t l i .
I t i l fi t t i l t t
iff tt t il t il fl t iti
l t t. A t t t i t ¶ l
t . i ti i f il
l f t f P ti i
t .

Fi :- I fl f l t t i li i l
t .
il i % t l/ % t . P ,
t i ; t l , t i ;3 t l ,
t i ; , i t l , t i ; t t l
, t i .

PRESSURE ROPOFTECO
 
UMN:-
ĭȘ
tM =
¨P ¨P = P

ti , t i i l it t
t ti ti . Alt i i i t
maximum attainable plate number, the generation of heat within the
column as a result of the work done in forcing the mobile phase through
the column at a very high pressure can seriously degrade column
performance. Pressures above 5000 psi ( 40 atm) do not appear
worthwhile for most HPLC separations.

PA
LEcAE
E
S
A
c APHASE 
The analytical performances improve dramatically when the particle
diameter is reduced, particularly when the column is operated at the
optimum velocity. Each time the particle diameter is halved, the
pressure drop required is raised by approximately a factor of four.
Often, however, the column length can be shortened significantly there
are no significantly. There are no practical operating conditions when
particles larger than 5 ëm would be desirable from the point of view of
achieving high plate numbers quickly. For a given column length, the
plate counts increased 1.7 times for a ë packing material compared
with a 5 ëm packing material. Furthermore, within limits, the plate count
is also directly proportional to the column length. Of course, one must
always keep in mind that the resolution of the two peaks is proportional
to only the square of the plate count. Eventually, however, the use of
ever finer particles or longer column of the same size particles requires
pressure that exceeds 5000 psi, the practical upper limit. Commercial
columns are available with packing materials with particle diameters of
, 5, and 10ë .

L LE 
H 
The ratio, L
§
dp is the number of particles to the column length
and is called as reduced length.
Then,

§
PO
TM
shows that when columns of the same reduced
length are eluted with the same eluent to give the same elution
time for an unretained solute, the same pressure drop is required. Finer
particles are usually paired with shorter columns to achieve a good
separation. This necessitates the use of higher inlet pressures to move
the mobile phase through the column at optimum velocity. Both effects
eventually results in serious technical difficulties. 

$cSSc
 
A solvent with low viscosity is always preferred in HPLC. While
maintaining constant pressure drop across the column, an increase in
velocity of the solvent always decreases the flow rate of the mobile
phase. The diffusion constant of the solutes ar e also affected by the
viscosity of the mobile phase.

APPLcA
c S
HPL
 HPLC is still in its infancy, but with the further development of new
support materials as well as new more sensitive detectors, it promises
to become more and more important. HPLC offers the advantages of
speed, resolution, and sensitivity it is especially useful for separating
the high molecular compound which have either a low vapour pressure
are under go pyrolysis when subjected to the heiger required
temperatures of gas chromatography .
The process have been applied to a wide variety of natural products
such as nucleic acid, urine, serum, carbohydrates, lipids, amino acids
bile acids and manufacture products such as pharmaceuticals,
pesticides, herbicides, surfactants, and antioxidants.

HPLc PHAAE
cALc S
cES

HPLC has wider application in the pharmaceutical industries for
drug analysis. There are some applications of HPLC in pharmaceutical
have shown below:-

%. HPLc cE
c
c A A
c
A
c 
HE
P Sc SAPLE
Each elute comes at a specific location, measured by the elapsed
time between the moment of injection [time zero] and the time when the
peak maximum elutes. By comparing each peak¶s retention time [t ]
with that of injected reference standards in the same chromatographic
system [same mobile and stationary phase], a chromatographer may
be able to identify each compound. 

igure %cdentification

In the chromatogram shown in Figure-1, the chromatographer knew

that, under these LC system conditions, the analyte, acrylamide, would
be separated and elute from the column at 2.85 minutes [retention
time]. Whenever a new sample, which happened to contain acrylamide,
was injected into the LC system under the same conditions, a peak
would be present at 2.85 minutes [see Sample B in Figure-2].
Once identity is established, the next piece of important
information is how much of each compound was present in the sample.
The chromatogram and the related data from the detector help us
calculate the concentration of each compound. The detector basically
responds to the concentration of the compound band as it passes
through the flow cell. The more concentrated it is, the stronger the
signal; this is seen as a greater peak height above the baseline.
 

igure cdentificationanduantitation

In Figure-2, chromatograms for Samples A and B, on the same

time scale, are stacked one above the other. The same volume of
sample was injected in both runs. Both chromatograms display a peak
at a retention time [t ] of 2.85 minutes, indicating that each sample
contains acrylamide. However, Sample A displays a much bigger peak
for acrylamide. The area under a peak [peak area count] is a measure
of the concentration of the compound it represents. This area value is
integrated and calculated automatically by the computer data station. In
this example, the peak for acrylamide in Sample A has 10 times the
area of that for Sample B. Using reference standards, it can be
determined that Sample A contains 10 picograms of acrylamide, which
is ten times the amount in Sample B [1 picogram]. Note there is another
peak [not identified] that elutes at 1.8 minutes in both samples. Since
the area counts for this peak in both samples are about the same, this
unknown compound may have the same concentration in both
samples.

.SEPAA
c 

HEE
AýLc
ES
$c
Ac S 
A  SALL PA
cLEScLcAL S 
The separation of all of the known metabolites of vitamin D2
and vitamin D found in biological fluids and the analysis of vitamin D
mixtures, purification of vitamin D metabolites, and identification of
radioactive peaks by applying HPLC. 
 Zorbax-SIL is a small-particle silica column packing that has
strong adsorptive affinity for the hydroxyl group(s) of vitamin D and its
metabolites.
 Figs.1 and 2 illustrate the resolution of vitamin D and its
metabolites. Although it is difficult to devise a single solvent system that
will elute 1,25-(OH)D in a convenient time and yet will resolve vitamin
D from the solvent front of the column, 10% isopropanol in Skellysolve
B (using 000 psi pressure) provides a reasonable compromise.
Obviously, an increase in the number of hydroxyl groups on the vitamin
D molecule increases the interaction with the silica adsorbent as
reflected by increased retention. The 1Į-hydroxyl groups apparently
interacts much more strongly with the silica than do the side-chain
hydroxyls. This is best illustrated by the retention of the dihydroxylated
1Į-OH-D(2or ) compounds over the trihydroxylated 24,25-(OH)D(2or )
compounds.
Thus, high-pressure liquid chromatography on silica allows for a
dramatic resolution of the naturally made1,25-(OH)2D and 25,26-
(OH)2D in normal lipid extracts, a resolution impossible on
conventional Sephadex LH-20 column chromatography or ordinary
silicic acid column chromatography. However, the interaction between
the side-chain hydroxyls and the silica is more than adequate to
provide an impressive separation of 24, 25-(OH)2D from 25,26-
(OH)2D and a separation of 25-OH-D from vitamin D .
 Of some importance is the resolution of vitamin D2 compounds
from vitamin D compounds. The silica columns do not permit the
resolution of vitamin D2 from vitamin D (Fig. ) or 1Į-OH-D2 from 1Į-
OH-D (Fig. 4), suggesting that the side chain without hydroxyls does
not interact significantly with the silica. However, the introduction of
hydroxyls on the side-chain positions of 24 or 25 permits a clear
resolution of the vitamin D2 and D analogs (Fig. and Fig. 5). A
partial separation of 24,25-(OH) & from 24,25-(OH)2D is also
achieved (Fig. 6). In all cases the D2 analog elutes before its
corresponding D analog. These results suggest that the methyl group
on (C-24 must shield or reduce the interaction of either the 24-OH or
the 25-OH, with the silica making such compounds less tightly held
than their D counterparts.
 Base-line resolution of vitamin D, 24-OH-D, and 25-OH-D is
achieved only by use of a less polar solvent system (2.5% isopropanol
is Skellysolve B), as depicted in Fig. . Again, side-chain hydroxylation
is necessary to provide a significant effect of the 24-methyl group on
the interaction with the silica adsorbent.

To illustrate the analytical usefulness of this system for biological

materials, Figs. 7 and 8 have been included. Fig.7 represents the
radioactivity and absorbance profiles of a blood plasma extract of
vitamin D-deficient rats given two 5-IU doses of 26,27- H-labeled 25-
OH-D 6 and 12 hr. before being killed. The extract was first
chromatographed on a Sephadex LH-20 column (1 X 60 cm) using a
solvent system of chloroform-Skellysolve B 65: 5, the 1,25-(OH)2D
region was combined with standard nonradioactive 25,26-(OH)2D and
1,25-(OH)2D compounds, and an aliquot was applied to the high-
pressure liquid column. Note that the presence of other tissue lipids did
not change the resolution or the elution position of the metabolites
appreciably.

Fig. 8 represents a profile from an extract of liver homogenate from

vitamin D-deficient chicks incubated with ~x-~H-labelevdi tamin D2
according to the procedure of Tucker, Gagnon, and Haussler and
prepurified on hydroxyalkoxypropyl Sephadex (1 X 60? cm; 10%
chloroform in Skellysolve B;). Aliquots of the 25-0H-D~region were then
chromatographed with marker vitamin D2,24-OH-D2, and 25-OH-D2.
Thus, the HPLC system is a powerful tool for the separation of all the
known metabolites of either vitamin D2 or vitamin D . There is a rough
correlation between the number of hydroxyl groups and elution position,
illustrating the more hydroxyls, the more tightly held is the compound.
However, the position of the hydroxyl on the molecule is also of great
importance. This is best illustrated by the fact that 1Į-OH-D (a
synthetic analog of 1,25-(OH)2D ), which is a dihydroxy compound, is
more tightly held than 24,25-(OH)2D , a trihydroxy compound. The
strong interaction of the 1Į-OH group undoubtedly is responsible for
the fact that 1Į,25-(OH)2D is held tightly to the column and elutes very
late in the profile. This interaction is also responsible for the impressive
and highly desirable separation of 25,26-(OH)2D from 1, 25-(OH)2D .

.SE
HPLc HcALSA ALScS
The necessity to analyse and purify chiral compounds has
increased. This has generated demand for chiral stationary phases
(CSP) with good chiral selectivity and high loadability. Kromasil
CelluCoat is a silica based CSP coated with tris-( ,5-dimethylphenyl)-
carbamoyl cellulose as the chiral selector. The chiral interaction
between analyte and a CSP like CelluCoat in normal phase mode is a
combination of steric, H-bond, ʌ±ʌ, and dipole interactions. Two of the
most predominant factors in analytical chiral HPLC are selectivity
leading to enantiomer separation, and hereafter the speed of the
separation. For analytical separations it is ideal to use small particle
CSP, which gives high chromatographic resolution and makes it
possible to run fast separations at high flow-rates without loosing
significant column efficiency. As demanded by the Food and Drug
Administration (FDA) chiral pharmaceuticals should be developed and
reach the market as single enantiomer products. This fact makes
robust chiral separation methods essential for product quality control
analysis, because the unwanted enantiomer is often the most critical
product impurity.
In the field of chiral chromatography preparative purification needs
range from semi-prep purification in the lab during early development to
full scale manufacturing of enantiopure drugs; e.g.

i) Atenolol 


Sample Name Atenolol 

Injection Volume 10 µl 
 Detector UV (2 5 nm) 
Mobile Phase CO2/MeOH + 0.2% IPHH (80:20 v/v) 
Temperature 40 °C
Flow Rate 4 ml/min
Column Name OD
Length 25 cm
Diameter 0.46 cm
Particle Size 10 µm
Observed Pressure 200 bar 


!. APPLcA
c  
 HPL c   c
c 
HE
PESS
AEA
c c S
HEScS 
 A facile method was established to enzymatically synthesize
rhapontigenin from the glycosylated parent compound rhaponticin. A
novel and simple high-performance liquid chromatographic method was
used for the determination of rhapontigenin prepared.

c

c  
Rhapontigenin, ( , ¶, 5 ±trihydroxy-4¶-methoxy-stilbene)
C15H16O4, MW: 258, is a stilbene found in Korean rhubarb rhizomes,
and is most abundant in the Rhei undulatum species. Rhaponticin, the
glycosylated parent compound of rhapontigenin has long been
employed as an oral haemostatic agent and to treat and prevent
allergies. Rhapontigenin, the aglycone of rhaponticin, has been
suggested to be the active molecule. Recent research has shown
rhapontigenin to be a
potent anti-allergic, anti-coagulant, and anti-

inflammatory compound. Rhapontigenin
±
also possesses potent anti -
cancer activity.
 

Structures of Stilbenes-
±




±
 ° 
  ±




 



ompound %    ! &

Rhapontigenin OH OH H OCH OH

Rhaponticin O- OH H OCH OH
Glucose

Synthesis of rhapontigenin from commercially available rhaponticin

is carried out. Furthermore, a selective, isocratic reversed-phase HPLC
method for the determination of rhapontigenin and its metabolites in rat
serum and its application to r ?
r and r ?
r
 kinetic studies is in
detailed.
HA
APHcSS
EA  c
c S
The HPLC system used was a Shimadzu HPLC (Kyoto, Japan),
consisting of an LC-10AT pump, a SIL-10AF auto injector, a
photodiode-array SPD-10A VP UV/VIS spectrophotometric detector
and an SCL-10A system controller. Injection volume was 150 ȝL.
The analytical column used was an amylose tris , 5-
dimethylphenylcarbamate (150 v 4.6 mm, ID, 5 Qm). The mobile phase
consisted of acetonitrile and 0.1% phosphoric acid ( 0:70, v/v), filtered
and degassed under reduced pressure prior to use. Separation was
carried out isocratically at ambient temperature, and a flow rate of 1.0
mL/min, with UV detection at 24 nm. Daidzein with methnol is used as
an internal standard.

E #A
cS
HEScS
HAP
cE c  
A 0.01M tetraethylammonium acetate buffer was made by adding
261 mg tetraethylammonium acetate to 100 mL HPLC water in a
volumetric flask. The pH was adjusted to 5.0 using 1M HCL. 4 mL
buffer was filtered and added to a clean glass test tube. 20 mg
rhaponticin was weighed carefully and added to the prepared buffer.
The rhaponticin solution was sonicated and vortexed until dissolved.
The rhaponticin solution was then placed in a 7 oC shaking water
bath. Next, an enzyme solution was prepared by adding 1 mL buffer to
6 mg ȕ-glucosidase. The enzyme solution was shaken gently and
directly added to the rhaponticin solution. The resulting solution was
incubated for 72 hours. 200 ȝL aliquots of the incubate were taken
every 24 hours and the reaction progression was monitored via HPLC.
t  ( ) '

100

 
i i g

0

  60 h a p o n tig e n in

% ' f P re

 40 h a p o n tic in
e

20

0
 
0 24 4 72 6

i  e ' f c   u   t i'   H ' u r  )

igure HPLC-monitored enzymatic reaction of rhaponticin into

rhapontigenin via ȕ-glucosidase.
&=E
Ec A
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HAP
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S$cAHPL 
The HPLC method has been applied to the determination of
rhapontigenin in pharmacokinetic studies in rats. Following
administration of rhapontigenin there was an apparent terminal
elimination half-life of ~6h for the parent compound.

!!
" /0123 453 3
t i'

r ti i
.
4 l6 c 6 r 2 3 i7 0 t 5 7
tr

+ !
- " 8 5 t 0 9 2 lit 5
,
+
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' + 

) !
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! #! ! $! %! !! #!
4 " "
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& '  * i( )

igure Mean intravenous pharmacokinetics of rhapontigenin i n male

Sprague Dawley rats.

One previously unidentified metabolite was detected with a

retention time of 4 minutes in the solvent front. The metabolite was
measured indirectly by treating samples with ȕ-glucuronidase and
measuring the increase in parent compound. The pharmacokinetics of
rhapontigenin appears to be qualitatively very similar to resveratrol in
the rat where a glucuronide metabolite is also present in plasma.

E
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 HAP
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 Lc$E cSES
 EA
E EA
c SS
E 
The HPLC method has been applied to the determination of
rhapontigenin and its metabolic products in the phase II metabolic
kinetic study of rhapontigenin in rat liver microsomes. Rhapontigenin
was added individually to microsomes in a concentration of 10 Qg/mL.
Following the incubation of rhapontigenin as parent drug at 7 rC in rat
liver microsomes with the UGT enzyme, a rapid and significant
decrease in rhapontigenin was detected. A metabolic peak was
observed, eluting at 4 minutes, which coul d not be resolved from the
solvent front.
 Due to the fact that the metabolite eluted with the solvent front,
the quantification of metabolite was determined indirectly using ȕ -
glucuronidase to hydrolyze the metabolite back to parent compound.
This technique has been extensively used in metabolism and
pharmacokinetic research to determine metabolite concentration over
time. ȕ-glucuronidase was added to a set of microsomal samples
instead of acetic acid/acetonitrile stop solution. These samples were
analyzed via HPLC along side of original microsomal samples exposed
to the stop solution. HPLC analysis confirmed the absence of the
glucuronidated metabolite. This same peak at the same retention time
was also apparent in the rat serum.
% cn creasin g  lu cu ro n id at

100
80
 et ab o lit e

Rhapontigenin
60
40 Glucuronidated
metabolite
20
0
0 10 20 0 40 50 60

im e  m in )


igure  Phase II Microsomal metabolism of Rhapontigenin in rat liver

microsome.

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4

 
 
The alkaloidal extract from A. ramiflorum has Anti-leishmanial
activity. For HPLC analysis, the crude extract was dissolved in
CH2Cl2:MeOH (80:20) and 10 µl were injected onto a Waters µ-
Bondapak RP-18 (reverse phase, 4.6 mm x 250 mm) column at 40 ˭.
Solvent A was 100 mmol/lit. ammonium formate in 0.12%
octanesulfonic acid (v/v)/, formic acid and acetonitrile (88:4:8, v/v),
while solvent B consisted of 100 mmol l -1 aqueous ammonium formate
containing 0.12% octanesulfonic acid (v/v)/formic acid/acetonitrile
(64:4: 2, v/v). The separation was carried out using a mixture of
solvent A and, a progressively increasing amount of B (0, 10, 40, 90,
100%) during 60 min. The flow rate was 1. ml min-1. The effluent was
monitored with a photodiode-array detector with windows at 222 nm
and 254 nm and also by mass spectral analysis of isolated eluates. ?
The major constituents of 4 ? r  alkaloidal extracts are:
ramiflorine A (1) and ramiflorine B (2), whose presence was monitored
by HPLC.

 
Fig. A: high performance liquid chromatography (HPLC)
chromatogram of standards mixture isolated from
4
r

?r ;
B: HPLC chromatogram of alkaloidal extract. Peaks - 1:
internal standard (tryptophol); 2: 10-methoxy-geissoschizol; :
ramiflorine A;4 : ramiflorine B.
Ö. c $ES
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HEE

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PEpH
The research is carried out to investigate some physicochemical
and solid-state properties of amoxicillin trihydrate (AMT) with different
powder pH within the pharmacopoeia-specified range. AMT batches
prepared using Dane salt method with the pH values from 4. 9 to 4.97
were subjected to further characterization studies. Optical and scanning
electron microscopy showed that different batches of AMT powders
were similar in crystal habit, but the length of the crystals increased as
the pH increased. Further solid-state investigations using powder x-ray
diffraction (PXRD) demonstrated the same PXRD pattern, but the
intensity of the peaks raised by the powder pH, indicated increased
crystallinity. Differential scanning calorimetry (DSC) studies further
confirmed that as the powder pH increased, the crystallinity and, hence,
thermal stability of AMT powders increased. Searching for the possible
cause of the variations in the solid state properties, HPLC analysis
showed that despite possessing the requirements of the United States
Pharmacopoeia (USP) for purity/impurity profile, there was a direct
relationship between the increase of the powder pH and the purity of
AMT, and also decrease in the impurity I (Į-Hydroxyphenylglycine)
concentration in AMT powder.

 
Amoxicillin trihydrate or AMT is a commonly used ȕ -lactam
antibiotic, which is highly active against a broad spectrum of bacteria.
High solubility, high rate of absorption, and stability of AMT under acid
conditions are among the most important advantages of this antibiotic.
 Different solid forms of the same chemical compound can exhibit
different physical and chemical properties including different solubility
and dissolution profiles, which in turn affect the bioavailability and
stability of the drug substance. For this study, the high-performance
liquid chromatography (HPLC) analysis was carried out for any possible
correlation between the solid state properties and the purity/impurity
profile of AMT batches with various powder pH.
The HPLC system consisted of a 616E pump, a 996 photodiode
array (PDA) detector, and a degasser module; data were acquired and
processed using a Millennium software Version 2.1 (all from Waters,
Milford, MA). The chromatographic separations were performed on
Spherisorb (Waters) C-18 columns (250 mm × 4.6 mm, with a particle
size of 5 ȝm). The mobile phase composition, column temperature, and
detector wavelength for determination of assay and related substance
were determined using the USP 25 method.

A ALScS 
 HPLC analysis was used to study the profile of the purity/impurities
within different samples. The profiles of the purity/impurities of different
AMT batches were investigated using standard USP 25 method. USP
has introduced several important impurities of AMT: A (6-
aminopenicillanic acid), B (L-amoxicillin), C (amoxicillin
diketopiperazines), D (penicilloic acids of amoxicillin), E (penilloic acids
of amoxicillin), F ( -(4-hydroxyphenyl) pyrazin-2-ol), and I (Į-
hydroxyphenylglycine). The results of HPLC analysis for AMT samples
with different powder pH showed that the samples had acceptable
purity/impurity profile (in the USP range), and the total percentage of
the impurities in each sample was less than 1%, which was comparable
to the standard AMT. The results showed that A, B, C, D, E, and F
impurities had no significant variations within different samples (with
powder pH from 4. 9 to 4.97). However, different samples showed
various concentrations of I (Į-Hydroxyphenylglycine) impurity. Of
interest, as the powder pH increased, the resulting chromatograph
showed a noticeable decrease in the impurity I peak intensity is shown
by arrows.


 

8.SEPAA
c 
Ac AcS 
Amino acids can be separated by HPLC by using cation±
exchange chromatography in which acids can be separated according
to their strengths on strong anion exchangers, with the weakest acids
emerging first, either by elution with a strong acid or, since acid
dissociation depends on pH, by gradient elution with buffers of
decreasing pH.
Amino acids, which add protons to form cations in the pH range
below their isoelectric points, can be separated on cation±exchangers
by gradient elution with buffers of increasing pH; here the more acidic
components emerge first and the most the most basic last.

ig Amino acid analysis; photometer senses the amino-acid-ninhydrin
complex (post column reaction) at 570 nm.

0. SEPAA
c  

SEcE SP  ELA
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SEcE
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LA
c  

 

urosemideMol.wt 0.75
Furosemide or frusemide is a loop diuretic used in the treatment
of congestive heart failure and edema. It is most commonly marketed
by Sanofi-Aventis under the brand name Lasix. It has also been used to
prevent thoroughbred and standard bred race horses from bleeding
through the nose during races. Along with some other diuretics,
Furosemide is also included on the World Anti-Doping Agency's
banned drug list due to its alleged use as a masking agent for other
drugs.

ethodonditions 
olumnCogent Bidentate C18Œ, 4ȝm, 100Å
atalog o. 40018-75P
imensions4.6 x 75 mm
obilephase70% Water, 0% THF 1% Acetic Acid

lowrate1.0 mL/minute
Peaks%. Furosemide
2. Related compound
cnjection$olume20 ȝL
etectionUV 254 nm

emperature25°C.

This active pharmaceutical ingredient, Furosemide USP and its

related compounds can be a difficult molecule to chromatograph with
conventional L1 (C18) columns due to silanol activity.
The excellent peak shape of Furosemide and its related compound-
A when used with HPLC; baseline resolution is achieved between
this specified impurity and Furosemide. The active is easily separated
from excipients in this tablet formulation.
%". SEPAA
c A E
E
c 
SL
 AcEc 
c
S
AýLE

LA
c 

 

Sulfonamides are used for the treatment of infections and
promotion of growth of livestock and fish. The residue of these
veterinary drugs in food is of serious concern, due to the risk to human
health (resistance to drugs and allergic or toxic reactions).
ethodonditions 
olumnCogent BidentateŒ, 4ȝm, 100Å
atalog o.: 40018-75P
imensions4.6 x 75 mm
obilephaseReverse Phase Gradient :
Solvent A: 100% DI water + 0.1% formic acid
Solvent B: 100% acetonitrile

ime%A%ý
0.00 70 0
0.00 ± 0.20 70 0
0.20 ± 5.00 0 100
5.00 ± 10.00 0 100
10.00 ± 10.01 70 0
10.01 ± 15.00 70 0

lowrate1.0 mL/min.
cnjection$olume10 ȝL
Peak1mg of the compound dissolved in 1 ml of substituted
sulfonamide (m/z 468) concentration for UV.
Analysis0.5 mg/mL and for LC/MS: 0.1 mg/mL
etectionA: UV 259 nm
B: LC/MS: Atmospheric Pressure
Chemical Ionization in positive mode:
APCI+, Single Ion Monitoring.
 

cSSSc  
The current sulfonamide detection methods are based on UV
absorption, but there is a need for methods detecting residues below
the maximum residue limits (MRL). LC/MS is a method of choice. A
simple and quick RP gradient was used to transfer the HPLC/UV
method to LC/MS for analysis of basic sulfonamides. APCI ionization
mode was more advantageous than ESI.

  LSc 
The wide applicability, speed, & sensitivity of HPLC have resulted
as a powerful tool for the-?
2 Quantitative/qualitative analyses of amino acids, nucleic acids,
proteins in physiological samples.
2 Measuring levels of active drugs, synthetic by-products, and
degradation products in pharmaceuticals.
2 Measuring levels of hazardous compounds such as pesticides
and insecticides.
2 Monitoring environmental samples.
2 Purifying compounds from mixtures.
Man is always worried about the purity of anything next to him.
Analysis is a science that gives the solution for. Convenience is great
boon of science, the convenience the method, the more it will be
popular. HPLC method is the method just heat the sample it will
ultimately say its story.