HPLC - High Performance Liquid Chromatography

HPLC stands for “High-performance liquidchromatography”(sometimes

referred to as High-pressure liquid chromatography)
High performance liquid chromatography is apowerful tool in analysis,
it yields high performanceand high speed compared to traditional
columnschromatography because of the forcibly pumpedmobile
phase.HPLC is a chromatographic technique that canseparate a mixture
of compoundsIt is used in biochemistry and analytical chemistry
toidentify, quantify and purify the individualcomponents of a mixture.
Chromatography: physical method inwhich separation of components
takesplace between two phases-a stationaryphase and a mobile phase
Stationary phase: The substance onwhich adsorption of the analyte
(thesubstance to be separated duringchromatography) takes place. It can
be asolid, a gel, or a solid liquid combination
Mobile phase: solvent which carries theanalyte (a liquid or a gas)
Chromatographic techniques are divided into differenttypes based on:
The type of chromatographic bed used
i.e. column chromatography (gas chromatography) andplanar
chromatography(paper and thin layer)
The physical state of mobile phase
i.e. gas chromatography and liquid chromatography
The separation mechanism
i.e. ion-exchange and size exclusion
HPLC is a type of liquid chromatography where thesample is forced
through a column that is packed with astationary phase composed of
irregularly or sphericallyshaped particles, a porous monolithic layer, or a
porousmembrane by a liquid (mobile phase) at high pressure.
COLUMN CHROMATOGRAPHY
Column chromatography involves the following:
1. Adsorption/retention of substance on stationary phase
2. Separation of adsorbed substance using mobile phase
3. Recovery of individual components by continuous flow of mobile
phase
4. Quantitative and qualitative analysis of solute and the components
which are recovered
•When distribution of solute is equal between two phases , theneffective
distribution Kd =1
•Individual zones adjacent to each other in the column wherethere is
sufficient space for equal distribution of solutes inbetween stationary
and mobile phase are called theoretical plates
• Length of theoretical plate in the column is called plate height
• More the theoretical plates, more efficient the separation
•2000 or more theoretical plates are ideal for a column
•Components which have more affinity towards mobile phaseelute first
PRINCILPE
To understand the principle of HPLC, we must first look at theprinciple
behind liquid chromatography
Liquid chromatography is a separation technique that involves:
•the placement (injection) of a small volume of liquid sampleinto a tube
packed with porous particles (stationary phase)
where individual components of the sample are transported along
thepacked tube (column) by a liquid moved by gravity.
The main principle of separation is adsorption
•When a mixture of components are introduced into the column.
variouschemical and/or physical interactions take place between the
samplemolecules and the particles of the column packing.
•They travel according to their relative affinities towards the
stationaryphase. The component which has more affinity towards
theadsorbent, travels slower. The component which has less affinity
towards the stationary phasetravels faster.
•Since no two components have the same affinity towards the
stationaryphase, the components are separated
HPLC is a separation technique that involves:
•the injection of a small volume of liquid sample into a tube packed
withtiny particles (3 to 5 micron (μm) in diameter called the stationary
phase)
•where individual components of the sample are moved down the
packedtube (column) with a liquid (mobile phase) forced through the
column byhigh pressure delivered by a pump.
•These components are separated from one another by the
columnpacking that involves various chemical and/or physical
interactionsbetween their molecules and the packing particles.
•These separated components are detected at the exit of this
tube(column) by a flow-through device (detector) that measures
theiramountThe output from the detector is called a liquid
chromatogramIn principle, LC and HPLC work the same way except the
speed, efficiency, sensitivity and ease of operation of HPLC is vastly
superior.
TYPES OF HPLC
I.BASED ON MODE OF SEPERATION
1.Normal phase chromatography - stationary phase is
polar(hydrophilic) and mobile face is non-polar (hydrophobic).
2.Reverse phase chromatography- stationary face is non-
polar(hydrophobic) and mobile face isPolar (hydrophilic).
•Polar-Polar bonds and Non Polar-Non Polar bonds have moreaffinity
than Polar-Non Polar bonds.
•Reverse phase chromatography is more commonly used as drugsare
usually hydrophilic
II.BASED ON PRINCIPLE OF SEPERATION
1.Absorption Chromatography
•In the Absorption Chromatography solute molecules bond directly to
thesurface of the stationary phase
•the component which has more affinity towards mobile phase elutesfirst
& the component which has less affinity towards stationary phaseelutes
later.No two components have same affinity towards mobile phase
&stationary phase.
2. Ion-exchange chromatography
•Ion exchange chromatography is a process that allows the separation
ofions and polar molecules based on their charge.
•It can be used for almost any kind of charged molecule including
largeproteins, small nucleotides and amino acids.
•Retention is based on the attraction between solute ions and
chargedsites bound to the stationary phase. Ions of the same charge are
excluded.
•The use of a resin (the stationary solid phase) is used to
covalentlyattach anions or cations onto it. Solute ions of the opposite
charge in themobile liquid phase are attracted to the resin by electrostatic
forces.
3.Ion-pair chromatography
•It is a form of chromatography in which ions in solution can be
"paired"or neutralized and separated as an ion pair on a reversed-phase
column.
•Ion-pairing agents are usually ionic compounds that contain
ahydrocarbon chain that imparts a certain hydrophobicity so that the
ionpair can be retained on a reversed-phase column.
4. gel permeation chromatography
•This type of chromatography lacks an attractive interaction between
thestationary phase and solute.
•The liquid or gaseous phase passes through a porous gel whichseparates
the molecules according to its size.
•The pores are normally small and exclude the larger solutemolecules,
but allows smaller molecules to enter the gel, causing them toflow
through a larger volume. This causes the larger molecules to passthrough
the column at a faster rate than the smaller ones.
5.Affinity Chromatography
•This is the most selective type of chromatography employed. It
utilizesthe specific interaction between one kind of solute molecule and
asecond molecule that is immobilized on a stationary phase.
For example, the immobilized molecule may be an antibody to
somespecific protein. When solute containing a mixture of proteins are
passedby this molecule, only the specific protein is reacted to this
antibody,binding it to the stationary phase. This protein is later extracted
bychanging the ionic strength or pH.
6.Chiral chromatography
•It involves the separation of stereoisomers. In the case ofenantiomers,
these have no chemical or physical differences apart frombeing three-
dimensional mirror images. Conventional chromatography orother
separation processes are incapable of separating them. To enablechiral
separations to take place, either the mobile phase or the stationaryphase
must themselves be made chiral, giving differing affinitiesbetween the
analytes.
III. BASED ON ELUTION TECHNIQUE
1.Isocratic elution
•A separation in which the mobile phase compositionremains constant
throughout the procedure is termedisocratic elution
•In isocratic elution, peak width increases withretention time linearly
with the number of theoreticalplates. This leads to the disadvantage that
late-elutingpeaks get very flat and broad.
• Best for simple separations • Often used in quality control applications
that support and are in close proximity to a manufacturing process
2. Gradient elution
•A separation in which the mobile phasecomposition is changed during
the separationprocess is described as a gradient elution
•Gradient elution decreases the retention of thelater-eluting components
so that they elutefaster, giving narrower peaks. This also improvesthe
peak shape and the peak height
• Best for the analysis of complex samples
• Often used in method development for unknown mixtures
• Linear gradients are most popular
IV.BASED ON SCALE OF OPERATION
1.Analytical HPLC
No recovery of individual components of substance
2.Preparative HPLC
Individual components of substance can be recovered
V.BASED ON TYPE OF ANALYSIS
1.Qualitative analysis
Analysis of a substance in order to ascertain the nature of itschemical
constituentsWe can separate individual components but cannot assess
thequantity in this analysis2.Quantitaive analysisDetermining the
amounts and proportions of its chemicalconstituents.Quantity of the
impurity and individual components can beassessed
INSTRUMENTATION
 A. Solvent delivery system(mobile phase)
•The mobile phase in HPLC refers to the solvent beingcontinuously
applied to the column or stationary phase
•The mobile phase acts as a carrier to the sample solution
•A sample solution is injected into the mobile phase of an assaythrough
the injector port
•As a sample solution flows through a column with the mobilephase,the
components of that solution migrate according to thenon-covalent
interaction of the compound with the column
•The chemical interaction of the mobile phase and sample, with
thecolumn , determine the degree of migration and separation
ofcomponents contained in the sample
•The solvents or mobile phases used must be passed through the
columnat high pressure at about 1000 to 3000 psi. this is because as the
particlesize of stationary phase is around 5-10µ, so the resistance to the
flow ofsolvent is high.
B.Pumps
•The role of the pump is to force a liquid (called the mobile
phase)through the liquid chromatograph at a specific flow rate,
expressed inmilliliters per min (mL/min).
•Normal flow rates in HPLC are in the 1-to 2-mL/min range.
•Typical pumps can reach pressures in the range of 6000-9000 psi (400-
to 600-bar).
•During the chromatographic experiment, a pump can deliver a
constantmobile phase composition (isocratic) or an increasing mobile
phasecomposition (gradient).
Types of HPLC pumps
There are several types of pumps used for HPLC analysis,
mostcommonly used are reciprocating piston pump,syringe pump
andconstant pressure pump
1.Reciprocating piston pumps:
•Consists of a small motor driven piston which moves rapidly back
andforth in a hydraulic chamber that may vary from 35-400µL in
volume
•On the back stroke, the separation column valve is closed, and thepiston
pulls in solvent from the mobile phase reservoir
•On the forward stroke,the pump pushes solvent out of the column
fromthe reservoir
•A wide range of flow rates can be attained by altering the piston
strokevolume during each cycle, or by altering the stroke frequency.
•Dual and triple head pump consists of identical pistonchamber units
which operate at 180 or 120 degrees out ofphase(this system is
significantly smoother because onepump is filling while the other is in
the delivery cycle.
2.Syringe type pump
•These are most suitable for small bore columns because this
pumpdelivers only a finite volume of mobile phase before it has to be
refilledThese pumps have a volume between 250 to 500mL
•The pump operates by a motorized lead screw that delivers mobilephase
to the column at a constant rate .The rate of solvent delivery iscontrolled
by changing the voltage on the motor.
3.Constant pressure pump
•In these types of pumps, the mobile phase is driven through the
columnwith the use of pressure from the gas cylinder
•A low-pressure gas source is needed to generate high liquid pressures
•The valving arrangement allows the rapid refill of the solvent
chamberwhose capacity is about 70mL
•This provides continuous phase flow rates
C. Injector
•The injector serves to introduce the liquid sample into the flow stream
ofthe mobile phase for analysis.
•It is equipped with six port valves so that a sample can be injected into
theflow path at continuous pressure
•For a manual injector, the knob is manually operated to deliver the
sampleto the column
•The knob is set to LOAD position for sample injection using a syringe,
the sample is injected into the sample loop, which is separated from
theflow path
•The knob is turned to INJECT position and the eluent travels through
theloop from the pump and delivers the sample to the column
•Typical sample volumes for manual injector are 5-to 20-
microliters(μL).
•The injector must also be able to withstand the high pressures of
theliquid system.
•An autosampler is the automatic version for when the user has
manysamples to analyze or when manual injection is not practical. It
cancontinuously Inject variable volume a of 1 μL – 1 mL
D.Column
•Considered the “heart of the chromatograph” the column’s
stationaryphase separates the sample components of interest using
various physicaland chemical parameters.
•It is usually made of stainless steel to withstand high pressure caused
bythe pump to move the mobile phase through the column packing
othermaterial include PEEK and glass
•The small particles inside the column are called the “packing”
whatcause the high back pressure at normal flow rates.
•Column packing is usually silica gel because of its particle shape,
surface properties, and pore structure give us a good separation
•Other material used include alumina, a polystyrene-divinyl
benzenesynthetic or an ion-exchange resin
– Pellicular particle: original, Spherical, nonporous beads,proteins and
large biomolecules separation (dp: 5 μm)
– Porous particle:common used,dp: 3 ~ 10 μm. Narrow sizedistribution,
porous microparticle coated with thin organic film
•The dimensions of the analytical column are usually-straight, Length(5
~ 25 cm), diameter of column(3 ~ 5 mm), diameter of particle(35μm).
Number (40 k ~ 70 k plates/m)
Guard column is used to remove particular matter andcontamination, it
protect the analytical column and contains similarpacking its
temperature is controlled at < 150 °C, 0.1 °CAs mention before ,
columns are divided into different typesaccording to their functions (see
types of HPLC)
E Detector
•The detector can detect the individual molecules that elute from
thecolumn and convert the data into an electrical signal
•A detector serves to measure the amount of those molecules
•The detector provides an output to a recorder or computer that results
inthe liquid chromatogram
•Detector is selected based on the analyte or the sample under detection
Commonly used detectors in HPLC
Ultraviolet (UV)
•This type of detector responds to substances that absorb light.
•The UV detector is mainly to separate and identify the principal
activecomponents of a mixture.
•UV detectors are the most versatile, having the best sensitivity
andlinearity.
•UV detectors cannot be used for testing substances that are low
inchromophores (colorless or virtually colorless) as they cannot
absorblight at low range.
•They are cost-effective and popular and are widely used in industry
Fluorescence
•This is a specific detector that senses only those substances that
emitlight. This detector is popular for trace analysis in environmental
science.
•As it is very sensitive, its response is only linear over a relativelylimited
concentration range. As there are not many elements thatfluoresce,
samples must be synthesized to make them detectable.
Mass Spectrometry
•The mass spectrometry detector coupled with HPLC is called HPLC-
MS. HPLC-MS is the most powerful detector,widely used
inpharmaceutical laboratories and research and development.
•The principal benefit of HPLC-MS is that it is capable of analyzing
andproviding molecular identity of a wide range of components.
Refractive Index (RI) Detection
The refractive index (RI) detector uses a monochromator and is one
ofthe least sensitive LC detectors.
•This detector is extremely useful for detecting those compounds that
arenon-ionic, do not absorb ultraviolet light and do not fluoresce.
•e.g. sugar, alcohol, fatty acid and polymers.
F Data processing unit (Computer)
•Frequently called the data system, the computer not only controls all
themodules of the HPLC instrument but it takes the signal from the
detectorand uses it to determine the time of elution (retention time) of
the samplecomponents (qualitative analysis) and the amount of sample
(quantitativeanalysis).
•The concentration of each detected component is calculated from
thearea or height of the corresponding peak and reported.

HPLC TROUBLE SHOOTING

Nano-Liquid Chromatography in Pharmaceutical and
Biomedical Research
Miniaturized separation techniques have emerged as environmentally friendly
alternatives to available separation methods. Nano-liquid chromatography (nano-
LC), microchip devices and nano-capillary electrophoresis are miniaturized
methods that minimize reagent consumption and waste generation. Furthermore,
the low levels of analytes, especially in biological samples, promote the search for
more highly sensitive techniques; coupled to mass spectrometry, nano-LC has
great potential to become an indispensable tool for routine analysis of
biomolecules. This short review presents the fundamental aspects of nano-LC
analytical instrumentation, discussing practical considerations and the primary
differences between miniaturized and conventional instrumentation. Some
theoretical aspects are discussed to better explain both the potential and the
principal limitations of nano-LC. Recent pharmaceutical and biomedical
applications of this separation technique are also presented to indicate the
satisfactory performance for complex matrices, especially for proteomic analysis,
that is obtained with nano-LC.
Introduction
The development of miniaturized systems is not a recent event. In the 1950s, the
use of capillary columns for gas chromatography was proposed by Golay , and
Horva´ th et al. used columns with small internal diameters (i.d.) for liquid
chromatography (LC) separations in the ‘60s. The nano-LC technique, as it is
currently known, was first introduced by Karlsson and Novotny in 1988, testing
packed columns with very small i.d. Recently, the great increase in miniaturized
LC systems has been driven by biological applications, primarily proteomics
research. Mixtures of proteins or peptides must be analyzed, and the quantities of
available samples and the low concentrations of the target analytes in the sample
matrix are not compatible with conventional LC systems. Traditional analyses by
high-performance liquid chromatography (HPLC) are performed using columns
with i.d. of 3.5– 4.6 mm. These analytical columns have typical flow rates of 1.0
mL/min. Columns with smaller dimensions (internal diameters of 20–100 mm) that
use flow rates of nanoliters per minute are called nano columns and are used in
nano-LC. Nano-LC is an alternative to conventional LC, providing more options
for chemical analysis. Virtually all samples analyzed by conventional LC can be
analyzed by a miniaturized technique. In this context, capillary electrophoresis and
capillary electrochromatography also complement and compete with nano-LC as
miniaturized liquid phase separations. Developments in nano-LC are tied to the
various advantages offered by this technique over conventional HPLC analyses.
Some positive aspects are:
(i) the large decrease in mobile and stationary phase consumption, including
toxic reagents;
(ii) the small sample needed;
(iii) the high efficiency separations while maintaining the same retention
behavior;
(iv) the easy coupling to mass spectrometry (MS). Currently, one of the most
important advantages is reduced waste generation, in accordance with the
 principles of green chemistry. However, the analytical instrumentation
used in nano-LC is still very expensive, limiting its widespread use.
Moreover, significant technical knowledge about nano-LC details is
required to prevent experimental difficulties, especially those related to
the instrumental arrangement. The number of publications focusing on
the applications of nano-LC has grown in recent years; however, neither
the theoretical aspects nor those related to instrumentation are reported in
these publications. Thus, this review covers the principal aspects of the
nano-LC technique and some recent pharmaceutical and biomedical
applications, especially in the most employed area, biological research.
Proteomic analysis, which corresponds to the major application of nano-
LC, is also presented.
Principles of Nano-LC
There is no agreement about the terminology of microscale LC. The terms
“microbore,” “microcolumn” and “capillary” LC are used interchangeably for
microcolumns of different i.d. According to Chervet et al. (9), separations
performed using columns of 0.50–1.0 mm i.d. are described as micro-LC;
columns of 100–500 mm i.d. are described as capillary-LC; finally, separations
using columns of 10–100 mm i.d. are described as nano-LC. This classification
includes separations in microchips because nano-HPLC columns on chips have
20 to 100 mm as the i.d. In this work, the classification of nano-LC will be used
for separations that operate at nanoliter flow rates, common for columns of 10–
100 mm i.d.
Theoretical aspects of nano-LC
During the chromatographic process, injected analytes can undergo dilution in the
column that alters separation efficiency. This dilution event, called
chromatographic dilution (D), is expressed by

where Co is the initial concentration and Cmax is the final concentration of the
analyte during the chromatographic process, dc is the column internal diameter, 1
is the total porosity of the column, L is the column length, Vinj is the sample
injectionvolume and k and H are the chromatographic parameters retention factor
and plate height, respectively. According to the equation, D decreases
proportionally with the reduction of the square of the column diameter. Compared
to conventional HPLC, the lower i.d. in nano-LC promotes a high reduction in D
value. Thus, downscaling of chromatographic systems means less chromatographic
dilution, increasing the mass detectability of the separation. The flow rate (F) in a
column is given by

where u is the linear velocity of the mobile phase. The reduction of dc leads to a
large reduction in the flow rate of the mobile phase, decreasing solvent
consumption and waste production in nano-LC separations. Theoretically, the
miniaturization of LC systems is very advantageous for liquid phase separations.
However, some practical separation aspects must be considered, because they
contribute to losses in separation efficiency.
Analytical instrumentation of nano-LC systems
In nano-LC, the conventional instrumentation is all miniaturized. Pumps,
connections, columns, injection loop and detection interface are dimensioned for
small volumes and low backpressure. These parameters can greatly influence the
chromatographic efficiency of nano-LC and need to be controlled for a successful
separation.
Pumps
Pumps for nano-LC need to present reproducible nano flow rates and stability
during the separation, and permit gradient elution at nano-scale levels. Two
primary systems can be used in nano-LC: split and splitless pumps, the latter being
commercially available. Split systems divide high flows (mL/min) from
conventional HPLC pumps by using a flow restrictor between the pump and the
miniaturized column. These systems allow for the use of the usual HPLC pumps
with an easily constructed nano flow restrictor. However, split systems may lead to
variable split ratios and low reproducibility of the nano flow, decreasing the
repeatability of the separation. Reproducible gradient elution is very difficult to
achieve, especially with homemade splitting devices: the different viscosities of the
mixed solvents can cause back-pressure fluctuations, limiting this elution mode.
Currently, splitless systems are widely used in nano-LC. These systems prevent
solvent losses and have more reproducible nano flow rates. Syringe pumps using a
single reservoir with a limited volume are better than split systems, but continuous
flow pumps, similar to conventional reciprocating pumps with two pistons per
channel, are currently the most widely used pump model. Continuous flow pumps
can be used in both isocratic and gradient elution at nano flows and adjustments of
the desired nano flow rates are easily achieved.
Tubing and connections
Peak broadening is a significant limitation to nano-LC development. The
dispersion of an analyte band in the column is described as a function of the i.d.
and length of the capillary. Thus, the lower the i.d. and length, the lower the on-
column dispersion contribution. Pre-column and post-column dead volumes may
lead to significant band broadening, which is critical when using columns with
reduced i.d. Inadequate tubing and connections increase band broadening, so that
the use of short, tight connections made with low volume tubing is required to
reduce this contribution to band broadening. Common connections are made of
stainless steel or polyetheretherketone; (PEEK) the latter is especially useful for
fused silica capillaries. Spaces formed by inadequate connections can also promote
lower separation resolution. Noga et al. reviewed some common limitations to
nano-LC separations, including problems with connections and their practical
resolution. They performed separations of bovine serum albumin (BSA) digests,
comparing both inadequate and adequate connections. Figure 1 shows the effect of
dead volume on the quality of the chromatographic separations. According to the
authors, inadequate fittings in the system cause a severe mixing inside the system
and no peaks were detected until 35 min.
Injection
The maximum injection volumes for nano columns can be expressed as a function
of the column length, plate number, retention factor or some other parameters, and
are generally a few nanoliters. Small injected volumes are a major problem in
nano-LC, causing loss of detectability, but larger injected volumes produce a band
broadening effect, decreasing the efficiency of the separation, especially for poorly
retained compounds. However, Heron et al. proved that, when using a weak
solvent for the sample, there is an enrichment effect and a gain in efficiency,
promoting the concentration of a sample plug after injection into a stronger mobile
phase. Commercial autosamplers, which usually work at microliter levels, require
an instrument adjustment for use in the nanoliter range. This may be overcome by
the use of a split valve between the injector and the column.
Nano-columns
Although columns of 10 mm i.d. can be employed, nano-LC columns of 75 mm
i.d. are the most frequently used. This i.d. column provides a good compromise
between detectability, loadability and robustness in nano-LC separations. In
general, nano-LC columns are made of polyimide-coated fused silica capillaries
that present flexibility, high mechanical resistance and a variety of internal
dimensions, but stainless steel and titanium tubes are also used for nano columns.
They can be packed with silica-based particles, filled by a monolithic bed or, less
commonly, wall-coated with appropriate organic or inorganic materials. The most
common particle sizes for packed nano columns are 3–5mm. However, particle-
filled small i.d. columns are difficult to prepare. Retention frits are required to
prevent the stationary phase from escaping; the preparation of the frits also
presents low reproducibility and often results in decreased efficiencies.
Nonhomogeneous beds after packing also reduce chromatographic performance.
Monolithic stationary phases are single rods of organic or inorganic material that
are produced inside the capillary column. No frits are required with monolithic
columns and the high porosity of these materials allows higher flow rates of mobile
phase, reducing the separation time. Monoliths can be prepared by using different
synthesis routes, organic or inorganic-based, and biocompatible materials are
interesting alternatives in biospecific analyses. Dolman et al. compared the
performances of packed and monolithic stationary phases for BSA separations and
evaluated the carryover effects in different capillary columns (Figure 2). BSAwas
chosen as a model for typical proteomic samples because it contains both
hydrophilic and hydrophobic peptides. Better separation efficiencies of the 11
peptides were attributed to the silica monolithic column, very similar to the
efficiency of the fused-core 2.7 mm silica packed column. The carryover effect,
which can compromise peptide analyses, was less with the fused-core 2.7 mm
silica than with the polymeric monolith and the porous 3 mm silica packed
columns also employed by the authors. The chemistry available for stationary
phases allows the applicability of nano-LC in a range of analyses. Reversed-phase,
hydrophilic interaction chromatography (HILIC), chiral selection, size exclusion,
ion exchange and other separation modes are applied to separations, according to
the target analytes. Many research groups prefer to prepare their own nano
columns specifically for their own purposes. A chiral stationary phase for nano-LC
was developed by Fanali et al. In this work, cellulose tris(3-chloro4-
methylphenylcarbamate)-coated silica particles were employed for the separation
of six neutral drugs, including thalidomide, a teratogenic drug. Enantiomeric
separations using a column of 100 mm i.d. were achieved in under 10 min and
were especially good for the separation of thalidomide, which cannot be
commercialized as a racemic mixture.

Detection
The types of detection for nano-LC are the same as those employed for HPLC
separations. Diode array detection (DAD) is commonly used in nano-LC, because
of its low cost, wide range of applicability and use of online detection. However,
due to the short path length of the nano column, detectability is limited when on-
column detection is applied. This is overcome by the use of specially configured
detection cells that provide longer light paths. Laser induced fluorescence and
inductivelycoupled plasma MS are also used in nano-LC detection, but these are
not robust enough to be applied for routine analysis. Biomedical and
pharmaceutical applications usually require good detectability and a universal
detection method, such as that provided by MS detection. The nano flow from the
column (frequently, 100–500 nL/min) is adequate for MS coupling through various
nanospray interfaces, especially electrospray ionization (ESI), which requires only
a small amount of eluent from the LC column to be successful.
Enrichment in nano-LC
Theoretically, the use of nano-LC promotes analyte enrichment more than
conventional HPLC. Reduced i.d. columns decrease chromatographic dilution and,
consequently, increase the instantaneous concentration of the injected analyte as it
passes through the components of the LC instrument. This enrichment factor is
attributed to lower dilution factors and is proportional to the square of the column
radius and the injected analyte volume, as discussed by Rieux et al. The smaller the
column radius, the lower the dilution factor, with a resulting increase in analyte
detectability. According to Cutillas, the use of a column of 75 mm i.d. can lead to
an analyte concentration factor of 5,000, compared to the use of a column of 4.6
mm i.d.. However, this enrichment factor is not easily achieved, because other
instrumental factors often decrease the observed analyte concentration, such as
excessive connection tubing, dead volumes from connections and disrupted nano
flow. Some experimental observations show that the small sample volumes
injected decrease the detectability of nano-LC compared to conventional HPLC,
especially when using ultraviolet (UV) detection. The application of MS detection,
multidimensional (nano)-LC or on-column trapping can greatly increase
detectability in nano-LC.
Hyphenation in nano-LC
MS is the most common nano-LC hyphenation. Coupling nano-LC to MS or
tandem mass spectrometry (MS-MS) has been applied in different areas, which has
solved various problems in the analytical sciences. For example, nano-LC
separations coupled to online MS (or, less commonly, offline MS) have increased
the diagnosis and treatment of several human diseases, promoting better quality of
life. Nano columns are also suitable for coupling to secondary separation
techniques, resulting in a two-dimensional (2D) chromatographic system. Luo et
al. proposed the 2D separation of a complex proteomic analysis from cervical
cancer cells using strong cation exchange and reversed-phase wall-coated nano
columns, coupled to MS detection. The authors concluded that the separation
capacity was improved by the orthogonality of the open tubular columns,
compared to one-dimensional reversed-phase.
Hyphenations also can be applied by using two miniaturized schemes, such as
biological microanalysis systems or coupling another (orthogonal) nano column in
the second dimension of the 2D separation. However, few reports of this kind of
hyphenation have been published to date, probably due to instrumental limitations.
Recent Pharmaceutical and Biomedical Applications of Nano-LC
Molecules of biological interest have to be quickly determined with highly reliable
results. In this context, recent advances in analytical instrumentation and sample
preparation methods have propelled biological analyses for the identification of
these interesting molecules. Nano-LC analyses are now applied for therapeutic and
veterinary drugs, doping control, disease diagnosis and the quantitative
determination of biomarkers and proteome identification; the latter are the
principal application fields, primarily because of the very low sample amount
required.
Proteomic research
Undoubtedly, proteomic studies respond to the major application of nano-LC
separations. Protein sequencing of complex biological samples is necessary for
biomarker identifications, disease control and clinical treatments, principally from
plasma and tissue samples. HPLC-based methods overcome the classical problems
of protein analysis, such as gel electrophoresis and immunoanalysis, which are
both limited by multiple steps before analyses. The diversity of proteome
complexity requires fast and unquestionable identification techniques, promoted by
the emergence of nano-LC coupled to MS and MS-MS. These have allowed the
exact determination of amino acid sequences from proteins or peptides, which is
assisted by a full identification database. However, classical methods are still used
with nano-LC–MS, because much information about protein sequencing and
peptide mapping is obtained by a combination of two or more identification
strategies. If not correctly diagnosed and treated, periodontitis can lead to acute
loss of teeth and systemic complications. Choi et al. proposed the identification of
the proteins of gingival samples from healthy and periodontitic patients while
searching for a specific biomarker for this inflammatory disease. The authors used
nano-LC–MS-MS for proteomic analysis and immunoassays for test confirmation;
305 proteins were identified in both sick and healthy patients and among these, 45
were directly related to periodontitis. Azurocidin was chosen as the best biomarker
and its levels were highly augmented in periodontitis patients, inhibiting osseous
differentiation in these cases. The principal conclusion of this work was to propose
the early diagnosis of periodontitis by the direct measurement of azurocidin levels
by nano-LC–MS-MS in complex oral samples, preventing the complications from
untreated disease. Proteomic analyses have been performed for synovial fluid from
rheumatic patients by using nano-LC–MS-MS. Osteoarthritis and rheumatoid
arthritis are both destructive articular diseases, characterized by a gradual
degradation of the cartilage tissues by defense cells, followed by
inflammationdisturbances. Mateos et al. identified peptides related to both articular
diseases and other peptides exclusive to each one. Knowledge of the proteome
from synovial fluids was important to detect protein fractions that acted as
biomarkers and promoted an efficient clinical control of patient treatments. Table I
lists other proteomic analyses conducted by nano-LC that have been reported in the
last two years
Biomarkers
Biomarkers are defined as endogenous indicators of a specific biological state,
usually a peptide or a carbohydrate. They can be experimentally measured and
evaluated for normal or disordered processes. In the biomedical sciences,
biomarkers are especially associated with healthy or diseased states. A biomarker
can also be a substance introduced into an organism to estimate its normal or
diseased function. Nano-LC plays an important role in biomarker analyses. The
low analyte concentration from biological samples requires
highly sensitive separation techniques and nano-LC coupled to MS or MS-MS
easily presents this characteristic. Garcı´a-Villalba et al. evaluated polyphenol
metabolism in human breast cancer cells using nano-LC–MS. The polyphenols
were found in extra virgin olive oil and their metabolites are proven to have anti-
tumor activity. The authors quantified the polyphenol metabolites according to
uptake time by the cancer cells and concluded that these biomarkers were easily
measured by nano-LC–MS. The search for brain trauma biomarkers in
cerebrospinal fluid was proposed by Sjo¨ din et al. They measured some proteins
that could indicate the level of brain trauma after a posttraumatic period by nano-
LC–MS-MS. To prevent protein degradation, the autosampler was kept at 108C.
The biomarkers were enriched and quantified by the use of a commercial ligand
over a wide dynamic range. However, even by using gradient elution, the
chromatographic run time was too long, probably due to the high interaction
between the stationary phase and the protein analytes.From human urine, a
biomarker of oxidative stress status, 8-isoprostaglandin F2a, was quantified for its
indication of some diseases, such as diabetes, cancer and Alzheimer’s disease. The
authors used a microchip-based nano-HPLC, and this system involved an
enrichment step before the chromatographic analysis. This enrichment promoted
an increase in the MS signal, proportional to an increase in the injected
concentration. The proposed method was validated and proved to be a sensitive
technique for isoprostaglandin analysis.
General drugs
LC is well established as an analysis tool for pharmaceutical targets in different
matrices. From drug discovery to quality assurance of drug formulations, validated
LC methods have been successfully employed by the pharmaceutical industry, in
research and development centers and for residual drug analysis in wastewaters.
Although nano-LC can be used instead of typical LC, the low acceptance of this
new technique at present is attributed to the high initial acquisition costs. However,
the use of nano-LC is slowly increasing because of the obvious reduction in the
volumes of required solvents and related waste disposal costs. Hsieh et al.
determined eight common penicillin dosages in pharmaceuticals. The authors also
determined these drugs in milk and tissue samples, proving the applicability of the
method in different biological matrices. A packed C18 column was prepared with
high repeatability for separation repetitions. Different polymeric frits were
evaluated for packing the
columns and polystyrene-based frits were chosen due to better separation
resolution than other tested polymer frits. For the simultaneous determination of
the penicillin compounds, the performance of nano-LC was compared by using
both UV and MS detection (Fig 3). The limit of detection (LOD) and limit of
quantification (LOQ) were higher with UV than with ion trap MS, as expected, but
the repeatability of peak areas from MS was lower than with UV detection.
Validation of the method using both detectors was conducted and residual
penicillin drug was found in some commercial tissue samples. D’Orazio et
al.performed the simultaneous determination of 18 sulfonamides, which are
antimicrobial agents used for human and animal therapies. The structurally similar
sulfa drugs were quantified by using nano-LC with UV and MS detection (Fig 4).
The multiresidual analysis was conducted in less than 40 min at a flow rate of 190
nL/min on a C18 core-shell column, which was chosen for having better
chromatographic resolution and separation efficiency than two other stationary
phase options. The authors concluded that both UV and MS detection presented
good detectability and the validation of the methods allowed their application in
residual sulfa drugs analysis from milk samples. Table II summarizes some other
applications of nano-LC in current pharmaceutical analyses.
Forensic analysis
The analyses of drugs of abuse and their metabolites in wastewaters can determine
the access of the population to thesesubstances and the public health requirements
for their control. Steroid hormones, hallucinogens, cannabinoids, opioids and
various prescription drugs are listed by US National Institutes on Drug Abuse as
commonly used drugs of abuse. Urine, sweat, blood (plasma) and saliva can be
analyzed for current drug use; however, hair appears to be the best specimen,
because it requires noninvasive sample collection. Compared to other specimens, a
hair sample has very little possibility of adulteration and informs a longer detection
period, revealing a history of drug abuse, if present (59). Hair specimens from
patients of a detoxification center were collected for the analysis of cocaine,
amphetamine, morphine and related drugs (59). The authors developed a simple
and validated nano-LC method as an alternative to inconclusive immunoassay
techniques, using special nanochip-LC instrumentation. They also significantly
reduced the sample preparation steps and the amount of sample required (less than
10% of usual quantity). Although it is an excellent tool for monitoring, nano-LC is
not usually applied for the identification and measurement of drugs of abuse,
probably due to the lack of nano-LC equipment in routine analysis laboratories.
Gas chromatography and conventional LC are the principal instrumental choices
because of their wide distribution in forensic centers, whereas immunoassay tests
are the most common analytical strategies for initial drug detection in biological
samples, due to their fast and easy execution.

Enzymes
Nano-LC is still not often used for enzyme analysis. Often, the stationary phase of
nano-LC alters enzyme conformations and their catalytic activity is reduced. Other
miniaturized techniques, such as capillary electrophoresis (CE), are preferred over
nano-LC, because they do not promote alteration of the real form of the enzyme.
However, reproducibility in nano-LC is higher than in CE, probably because the
pressurized flow is more stable than the electroosmotic flow generated inside the
capillary in CE. Krˇı´zˇ ek and Kubı´cˇ kova´ reviewed the most recent methods
for kinetic enzyme assays and showed that CE and its modes of separation were
widely used for enzyme analysis, whereas nano-LC was used in only a few papers
in recent years. One possibility to overcome the limitations of enzyme analysis in
nano-LC is the use of bioaffinity columns. These special particulate or monolithic
stationary phases immobilize the enzyme in an accessible conformation without a
significant loss of the original enzymatic activity. According to Tetala and van
Beek, bioaffinity columns for nano-LC can easily be prepared from organic or
inorganic-based materials, not only for enzymes but also for other biomolecular
analyses related to the immobilized enzymes.
Conclusions and Outlook
Today, the miniaturization of analytical instrumentation presents an important role
in the development of analytical sciences, which is encouraged by studies in many
different areas. Methodologies for pharmaceutical and biomedical applications
must be sensitive enough to detect and quantifybiologically relevant substances
present in minute quantities. Especially for these low-concentration substances, the
employed techniques must have excellent detectability and unquestionable
identification, as provided by nano-LC–MS and nano-LC– MS-MS hyphenations.
The principal limitation at the present to wider use of nano-LC is the high cost of
the analytical instrumentation. However, the rapid development of new equipment
is overcoming this limitation, expanding nano-LC to routine laboratories and
industries. The chemistry of commercially available columns for nano-LC is also
still a limiting factor compared to the many and versatile conventional LC
columns, which cover a wide range of analytical possibilities. Stationary phase
preparation, focusing on new nano columns such as monolithic and sub-2 mm
particulate separation columns, is still a field that is only initiating its development.
In the near future, however, nano-LC has the potential to reach a consolidated
position in the analysis of biological molecules as a complement to electrophoresis
and immunoassays.

Preparative HPLC
Preparative HPLC
Introduction
 Chromatography is a technique of separating mixture of substances into their
components based on their composition and molecular structure.
 The process involves moment of sample between two phases.
 The separation of various components takes place depending upon affinity
of various molecules towards stationary mobilephase. Hplc uses this
technique and the mobile phase is pumped through stationary phase at high
pressure.
PREPARATIVE HPLC:
 It is a technique used for the isolation and purification of varitey of
chemicals, pharmaceutical compounds and biological molecules.
 Types of hplc based on the scale of operation
1.Analytical hplc
2.preparative
 Mainly in preparative hplc sample goes from detector into fraction collector.
PRINCIPLE:
 Adsorption
 Similar to hplc
 The only difference is sample goes from detector into fraction collector.
INSTRUMENTATION:
 Solvent reservoir
 Pump
 Preparative injector
 Detectors
 Programmers
 Recycle valve
 Fraction collector
SOLVENT RESERVOIR: Material of construction glass or stainless steel
 For biologically sensitive or liable substance coating of biocompatible
material.
  PREPARATIVE PUMP: Requires high eluent flow rate 10 and 100 ml/min
and large internal diameter of columns.
 A large pistonhead Is required to work at flows 10-100ml/min.
PREPARATIVE INJECTOR:
 Should inject samples with in the rate of 0.1-100 ml
 Reodyne injector is used.
PREPARATIVE COLUMNS:
 Column is the heart of liquid chromatography
 Sample distribution plate is used to distribute the sample across the column
 It depends on the particle size, scale of separation and on the nature of the
material to be separated,there are two types of column packings
 Particle size more than 10mm-dry packing
 Particle size less than 20mm- slurry packing
PREPRATIVE DETECTORS:
 In preparative hplc eluents are diluted with more mobile phase and then
passed through the detector
 Detectors are same as hplc
FRACTION COLLECTOR:
 In preparative hplc sample goes from detector to fraction collector
 The fraction collector diverts the flow either to waste or, to a fraction
container via fraction collection needle which can achieve by using diverter
valve.
FRACTION COLLECTION METHODS :
1.Manual fraction collection-highest flexibility based onsingle plot.
2.Peak based fraction collector-based upon detector response.
3.Mass based fraction collector-compounds with the desired mass is selectively
collected.
4.Time based fraction collector-based on time of interval.
INSTRUMENTION
 1. Solvent reservoir
2. Pump
3. Preparative injector
4. Preparative column
5. Detector
6. Programmer
7. Recycle valve
8. Fraction collector

APPLICATIONS OF PREPARATIVE HPLC:

• Purification in medicinal or high through put chemistry.
 • Purification in natural product chemistry.
• Purification of by products for impurity analysis.
• Recovery collection
PHRAMCEUTICAL APPLICATIONSOF HPLC:
• It includes resolution, identification and quantification of a compound it also
aids in chemical separation also and purification the other applications
includes.
• To control drug stability
• Tablet dissolution study of pharmaceutical dosage form
• Pharmaceutical quality control.
ENVIRONMENTAL APPLICATIONS:
• Detection of phenolic compounds in drinking water
• Bio monitoring of pollutants.
APPLICATIONS IN FORENSICS:
• Quantification of drugs in biological samples.
• Identification of steroids in blood, urine etc.
• Determination of cocaine and other drugs in blood.
APPLICATIONS OF CLINICAL TESTS:
• Urine analysis, antibiotics analysis in blood
• Analysis of bilirubin in hepatic disorder
• Detection of endogenous neuropeptides in extracellular fluid or brain etc.,

Chiral Separation of Pharmaceuticals by High Performance Liquid

Chromatography
INTRODUCTION Molecular chirality is a fundamental consideration in drug
discovery, it is very necessary to understand and describe biological targets as well
as to design effective pharmaceutical agents. A visual context was ever provided to
position chiral technology in the highly complex and evolving drug discovery
environment with its numerous challenges, hurdles, and tools (see Fig. (1))

Nowadays, chiral separation covers an important area ofanalytical chemistry of

relevance to a wide variety of scientific professionals. Many enantiomeric forms of
drugs have been recognized to have different physiological and therapeutic effects.
Very often, only one form is pharmacologically active in an enantiomeric pair. It
is, thus, hardly surprising that the pharmaceutical industry needs effective methods
to determine enantiomeric purity and obtain effective and safe drugs with single
steroconfiguration . Ward et al. ever reviewed various developments and
applications in chiral separations. A large number of approaches have been used to
isolate enantiomers (such as gas chromatography, thin-layer liquid
chromatography, capillary electrophoresis, liquid chromatography, supercritical
fluid chromatography, electrokinetic chromatography, etc.), among the approaches
enantioselective liquid chromatography has become the most popular technique for
quickly obtaining limited quantities (from mg to multi-grams) of pure single
enantiomers, especially in drug discovery. Moreover, HPLC is well recognized as
a powerful, fast, highly efficient and selective technique, successfully employed
for preparation and analysis of enantiomers of drugs. Okamoto et al.reported about
chiral HPLC for efficient resolution of enantiomers and mentioned that more than
half of the determinations have been carried out by chiral HPLC in all the relative
reports. In recent years, some new HPLC methods have been developed for the
analytical and semipreparative resolution of new drug enantiomers. Although
HPLC may be considered as a mature technology, many advances have been made
to improve the separation capability and the efficiency of method development and
sample analysis continuously. Ever increasing demands for chiral analysis and for
higher laboratory efficiency have driven the development
of various options for high-speed analysis with the desire to improve chiral
resolution. HPLC can be used either indirectly with chiral derivatization reagents
(CDR) or directly with chiral stationary phases (CSPs) or chiral mobile phase
additives (CMPA) to separate chiral compounds. The characteristics of these three
chromatographic methods are summarized in Table 1. At present, large kinds of
new materials have been developed as chiral stationary phases (CSPs) in liquid
chromatography. Also, some new chiral derivatization reagents and new chiral
mobile phase additives have been used for enantiomeric separation successfully.
Moreover, large amounts of applications of several hyphenated techniques (LC-
MS, LC-CD and LC-OR, etc.) were reported succesively in chiral analysis . For
example, when an enantiomer expresses exciton coupling that is observed in the
CD spectrum, the absolute stereoconfiguration can be assessed in combination with
molecular modeling studies of the lowest energy conformers. This review focuses
on the current developments in column technology and method development
approaches, then a brief discussion of advances is given in hyphenated techniques.
Related methods of rapid analysis include micro-columns, small particle columns
and multidimensional chiral high performance liquid chromatography.
2. NOVEL CHIRAL STATIONARY PHASES (CSPS)
As is well known, chiral separation by HPLC with CSPs has become a powerful
tool in the development of chiral drugs. The HPLC-CSPs demonstrated great
success for performing chiral separation in pharmaceutical industry, because the
HPLC can be used both for analytical or preparative purpose in a very efficient and
flexible way. So far, there are numerous commercially available chiral selectors or
CSPs for the resolution of racemic drugs. Several new selectors for HPLC
applications have been developed in recent years,such as cyclodextrin,
polysaccharide, protein, macrocyclic antibiotics, crown ethers, etc. Table 2
presents various typical kinds of chiral selectors, including their characterizations,
structures and possible separation analytes.
2.1. Brush-type CSP
Brush-type CSPs, also called ‘Pirkle CSPs’, were invented by Pirkle’s group since
the early 1980s (1980, 1991). The chemical structural characteristics of this type of
CSPs are based on the presence of π-donor groups, π-acceptors group or the groups
that could form multiple hydrogen bonds, and these groups can give a good fit to
the “three point interaction principle” theory. This type of CSPs (such as DNB-
Leu, DNB-PG, Whelk-O 1, Whelk-O 2, ULMO, α-Burke, β-Gem 1, all from Regis
Technologies, Inc. USA) have been extensively used for chiral analysis. Some
structural improvement of existing brush-type CSP have been carried out for
improving limited separating capability of the CSPs. These novel CSPs show
strong separating capability for many specific chiral compounds. Landek et
al.adopted brush type chiral stationary phases derived from L-leucine for
enantiomeric separation of 1,1’-binaphthyl-2,2’-diol enantiomers. A novel chiral
packing material for HPLC was prepared by connecting (R)-1-phenyl-2-(4-
methylphenyl) ethylamine (PTE) amide derivative of (S)-isoleucine to
aminopropyl silica gel through 2-amino-3, 5-dinitro-1-carboxamido-benzene unit.
A Pirkle-type chiral stationary phase for column chromatography based on N-p-
Amino benzoyl-L-glutamic acid has been synthesized for resolution of β-
methylphenylethylamine
2.2. Cyclodextrin CSP
The first CSP containing cyclodextrin (CD) bonded to silica gel was developed by
Armstrong and DeMond in 1984. Recently, both the native CD CSPs and the
modified CD CSPs with improved enantioseparation performances have been used
in many cases. Berkecz et al. employed a kind of β-CD based chiral stationary
phase for enantioseparation of 1-(α-aminobenzyl)-2-naphthol and 2-(α-
aminobenzyl)-1-naphthol analogs. Despite the numerous CD derivatives were
developed as chiral selectors in the past decades for the purpose of chiral
separation, a great need still exists for further effort to synthesize novel CD-CSPs
with improved chiral recognition ability towards a large pool of racemic analytes
with shorter time and greater accuracy. Tang et al. provided a meaningful overview
of recent work carried out in their laboratory on the synthesis of four series of
mono-substituted positively charged CDs which were applied as chiral selectors in
the separation of derivatized amino acids, anionic pharmaceutics and neutral
analytes by
and neutral analytes by chromatographic techniques. These novel cyclodextrin
CSPs showed good chiral recognition ability in actual separation.
2.3. Polysaccharide CSP
Polysaccharide type CSPs have proven to be one of the most useful CSPs because
of their versatility, durability, and loadability. In recent years, a broad range of
commercially available polysaccharide phases have been produced by Daicel
group (Osaka, Japan), based on cellulose, amylose esters or carbamates. The
cellulose-based columns (e.g. Chiralcel OD) and the amylose-based columns (e.g.
Chiralcel AD) are frequently used for the enantioseparation .Immobilised
polysaccharide-based CSPs are a new generation of chiral chromatographic
materials combining the remarkable enantioselective performance of the
polysaccharide derivatives with solvent versatility in enantiomeric resolution.
Since 2004 three immobilised polysaccharide-derived CSPs have become
commercially available: Chiralpak IA, Chiralpak IB andChiralpak IC by Daicel
Chemical Industries, Ltd. (Tokyo, Japan). Zhang et al.employedChiralpak IC for
separation of 70 chiral molecules. The results indicated that Chiralpak IC exhibited
high enantioselectivity for a large number of enantiomers. In addition, the
versatility of mobile phase is an important factor for chiral separation on Chiralpak
column. The chromatographic performances of Chiralpak IA under various mobile
phases were investigated. It was found that four solvents (methyl tert-butyl ether,
dichlormethane, tetrahydrofuran and ethyl acetate) were particularly versatile in
mobile phase. So it was highly recommended to screen these solvents first for
enantioselectivity. Jin et al. compared several polysaccharide-derived chiral
stationary phases for the enantiomeric separation of N-
fluorenylmethoxycarbonylα-amino acids and N-protected α-amino acids. The
result indicated that all the CSPs resulted in the greatest separation factor.
However, Chiralpak AD and Chiralcel OD of the typical coated-type CSPs are not
compatible with all solvents of the mobile phase (normal phase mode is mainly
used), because the chiral selectors of polysaccharide derivatives for these CSPs are
coated on a silica matrix. Moreover, owing to the compatibility with a wide range
of solvents of the covalently
bonded CSPs, it is expected some new application of Chiralpak IA and IB in
enantiomer separation, especially for preparative purpose. Some literatures
reported the technique for immobilizing the polysaccharide derivatives onto the
silica gel formerly.
2.4. Protein-based CSP
The advantages of protein-based CSP include enantioselectivity to a wide range of
compounds because all proteins have certain ability to discriminate chiral
molecules. CSPs based on bovine serum albumin (BSA), human serum albumin
(HAS), α1-acid glycoprotein (AGP), ovomucoid from chicken egg whites
(OMCHI), avidin (AVI), cellobiohydrolase I (CBH I) and pepsin are now
commercially available. So far, many protein-based CSPs have been developed,
and some studies were published on the mechanisms and applications of
enantioseparation of chiral drugs. In recent years, serum albumin of other species,
penicillin G-acylase, antibodies, fatty acid binding protein and streptavidin were
newly introduced as the chiral protein-based selectors in CSPs. Fig. (2) shows
good enantioseparation of tryptophan, warfarin and ibuprofen onHSA columns
prepared by N-(4-carboxycyclohexylmethyl) maleimide (SMCC) and succinimidyl
iodoacetate (SIA) methods [58]. The chiral recognition mechanisms of these
proteins were persumed by molecular modeling, ligand docking, and X-ray
crystallography, which can effectively promote the rapid development in designing
new protein-based CSPs with specific group proteins used for chiral selectors.
2.5. Macrocyclic Antibiotics CSP
Macrocyclic antibiotics used as chiral selectors were first introduced by
Armstrong’s groupand represent a relatively new class of chiral selectors in
separation science. The selectors have a large number of stereogenic centers and
functional groups, which could realize multiple interactions with chiral molecules.
So far, several macrocyclic antibiotics (including rifamycins B and SV, avoparcin,
teicoplanin, ristocetin A and vancomycin) have been used as chiral selectors
widely . A vancomycin degradation product-based CSP has been adopted for chiral
separation of racemic compounds. Honetschlagerova-Vadinska et al.made
satisfying evaluation of the enantioselective performance of two teicoplanin-based
CSPs Chirobiotic T and Chirobiotic T2, and then the authors discussed the possible
interaction mechanisms on the basis of the separation results and some theories.
Pataj et al. compared three teicoplanin-derived CSPs (Chirobiotic T, T2 and TAG)
for the enantioseparation of β2-homoamino acids. The result indicated that
Chirobiotic TAG, whose structure contain no sugar units, may promote the
interactions of the enantiomers of β2-homoamino acids with branched alkyl or aryl
side-chains, whereas β2-homoamino acids with alkyl side-chains Chirobiotic T and
T2 seem to be more favorable. Vancomycin-CSP was used for determinating
thalidomide enantiomers in biological samples.
2.6. Other CSPs
Chiral synthetic polymers, crown ethers, chiral imprinted polymers, ionic
compounds, chiral ionic liquid, and some natural products have also been used as
CSPs for the enantioseparation of pharmaceuticals and their unique structure
always result in uncontemplated separation ability for chiral drugs. A
comprehensive review on the synthesis and application of chiral synthetic
polymers was reported by Nakano. Two new synthetic polymeric CSPs based on
trans-(1S, 2S)-cyclohexanedicarboxylic acid bis-4-vinylphenylamide and trans-
N,N-(1R,2R)-cyclohexanediyl-bis-4-ethenylbenzamide monomers were prepared
and evaluated by HPLC in normal phase system . Further, a new synthetic
polymeric CSP for liquid chromatography was prepared via freeradical-initiated
polymerization of trans-9,10-dihydro-9, 10-ethano-anthracene- (11S,12S)-11,12-
dicarboxylic acid bis-4-vinylphenylamide. This new polymeric CSP showed
special enantioselectivity for many chiral compounds in multiple mobile phases.
Crown ethers are macrocyclic polyethers that can form host-guest complexes. The
CSPs of crown ethers have an
excellent chiral recognition ability and are easily bound covalently to the support.
Berkecz et al. employed a chiral stationary phase containing (+)-(18-crown-6)-
2,3,11, 12-tetracarboxylic acid for chiral separation of several β-amino acids. A
novel CSP prepared by immobilizing a chiral pseudo-18-crown-6-type host onto 3-
aminopropyl silica gel was used for the enantioseparation of 18 common natural
amino acids efficiently. In addition, a CSP based on (-)-(18-crown-6)-2,3,11,12-
tetracarboxylic acid was evaluated for the direct resolution of the enantiomers of
dipeptides and tripeptides. Molecularly imprinted polymers (MIP) have been
exploited as CSPs since the pioneering work of Wulff in the 1970s. Several
excellent reviews were given in past years on the use of molecularly imprinted
stationary phases in HPLC. Molecularly imprinted polymers CSPs can be
extremely successful in most chiral for many certain compounds. CSPs with
ionizable groups have been developed in past few years. Hoffmann et
al.investigated the synergistic effects on enantioselectivity of zwitterionic CSPs for
the separation of chiral acids, bases, and amino acids. For example, a novel strong
cation-exchange-type of chiral stationary phase has been used for separation of
cinchona alkaloids. Ionic liquids (ILs) are a group of organic salts that can keep
liquid-state at room temperature. They have unique chemical and physical
properties, including air- and moisture-stable, a high dissolving capacity and
virtually no vapor pressure. Because of these properties, they can serve as “green”
recyclable alternatives to the volatile organic compounds which are traditionally
used as industrial solvents. Advances in ILs have made the synthesis of chiral ILs
become a subject of intense study in recent years. The popularity stems from the
fact that it is possible to use chiral ILs as a chiral stationary phase in
chromatography for the determination of pharmaceutical compounds. Tran et al.
successfully synthesized both enantiomers of a novel chiral ionic liquid, (R)- or
(S)-[(3-chloro-2-hydroxypropyl) trimethylammonium] [bis((trifluoromethyl)-
sulfonyl) amide] ((R)- and (S)-[CHTA]+[Tf2N]-) in optically pure form which are
commercially available by simple ion exchange reaction from corresponding
chloride salts, and they successfully developed a novel method based on the near-
infrared technique with this chiral IL serving both as solvent and as a chiral
selector for the determination of enantiomeric purity. Yu et al.also synthesized a
series of structurally novel chiral ionic liquids with an either chiral cation or chiral
anion, or both. Structurally novel and strong intra- and intermolecular chiral
recognition ability of these chiral ILs showed that they could be used in a variety
of applications including as chiral solvents for asymmetric synthesis and as CSPs
for chromatographic separation. In addition, a large number of natural products
have been used as novel chiral stationary phases. A novel chiral stationary phase
based on the L-RNA aptamer was a kind of highly biostable stationary phase due
to the intrinsic insensitivity of L-RNA to the RNase degradation. Such an
approachallowed one to reverse the elution order of enantiomer relative to that
obtained with the corresponding D-RNA CSP. Wang et al. adopted glucose,
cellobiose, lactose and raffinose as chiral stationary phases in HPLC, and the novel
chiral stationary phases also possess high enantioseparation selectivity, which may
be used in normal-phase and reverse-phase mode, and there is a great chiral
discriminating complementary to present study.
3. CHIRAL DERIVATIZATION REAGENTS AND CHIRAL MOBILE
PHASEADDITIVES
3.1. Chiral Derivatization Reagents (CDRs)
The indirect method involving the derivatization process with some suitable chiral
tagging reagent is an efficient way for the separation of many enantiomers,
especially when suitable CSPs were not available. The mechanism of indirect
separation is based on the use of chiral derivatization reagents to form a complex
of diastereomers, the diastereomers differing in their physicochemical properties
can hence generally be separated by conventional chromatographic columns. So
far, enormous chiral derivatization agents are available. A review described the
application of chiral derivatization agents in the HPLC separation of amino acid
enantiomers. A new chiral derivatization agent, 2,3,4,6-tetra-O-acetyl-β-D-galacto-
pyranosyl isothiocyanate (GATC) was adopted for chiral separation of several β-
blockers. Ten novel CDRs have now been used for HPLC enantioresolution of
some β-amino alcohols. The method was also found successful for the separation
of 20 diastereomers from a mixture. Kurosu et al. reported unprecedented chiral
derivatizing agents that allowed for the determination of the absolute
configurations of a wide range of amino acids by only analyzing the carbamate
nitrogen protons in 1H NMR spectra. Bhushan et al. synthesized chiral hydrazine
reagents for enantioseparation of carbonyl compounds. Another new chiral
derivatizing reagent synthesized from s-triazine chloride was employed for
enantioseparation of α-amino acids by reversed-phase liquid chromatography.
With more and more new chiral derivatizing agents appear for selection, the
methodology of chiral derivatizing agents will play a more important role in chiral
separation.
3.2. Chrial Mobile Phase Additives (CMPAs)
It is possible to effect an enantiomeric separation using conventional HPLC
stationary phases by adding a chiral selector in the mobile phase. In this method,
enantiomeric separation is accomplished by the formation of a pair of transient
diastereomeric complexes between racemic analyte and the chiral mobile phase
additive. The advantages of this technique are so many, the most obvious of them
are simplicity and flexibility; moreover there is no need for chiral derivatization.
The frequently used CMPAs are ion-pairing reagents, ligand-exchanging reagents,
protein-affinity reagents and cyclodextrin inclusion reagents. Among them,
cyclodextrin was used for chrial mobile phase additives generally. Ma et al.
employed sulfated β-cyclodextrin (S-β-CD) as CMPA for enantiomeric separation
of chiral amine and explored the chiral recognition. The results showed that S-β-
CD could undergo a conformational change with increasing temperature.
Particularly, the aromatic part of the molecule was included inside the cyclodextrin
through hydrophobic interactions with the interior of the macrocycle, which could
reflect the interactions between the CMPA and analytes. Moreover,
norvancomycin and vancomycin were adopted as CMPAs in chiral separation and
gained expected affect for several certain analytes.
4. HYPHENATED TECHNIQUES
4.1. LC-MS
During the last decade, separation techniques coupled with mass spectrometry
became an essential and powerful tool in molecular biochemistry, especially in the
analysis of chiral compounds of pharmaceutical, clinical, environmental and/or
agro-chemical interests. In order to evaluate the pharmacokinetics of a single
enantiomeror of enantiomer mixture, manufacturers should develop quantitative
assay for the individual enantiomers in vivo samples early in the drug development
process. Currently, there is a trend toward converting chiral HPLC methodologies
into more sensitive mass spectrometry-based approaches without losing the
enantioselectivity of the assay. The methodology of chiral LC-MS is becoming
increasingly important for the routine analysis of chiral drug and their metabolites
in biological matrices, also for toxicology research. The recent developments in
chiral liquid chromatography coupled with atmospheric pressure ionization tandem
mass spectrometry (LC-API-MS/MS) for the analysis of pharmaceuticals were
reviewed by Chen et al. Various new ion sources including electrospray ionization
(ESI), atmospheric pressure chemical ionization (APCI) and atmospheric
photoionization (APPI) have been employed for chiral separation of
pharmaceuticals. Among these soft ionization techniques, only APPI source is
compatible with both reversed phase and normal phase conditions without the
safety concerns of a potential explosion. The schematic of APPI with a chiral
column for the pharmaceuticals is shown in Fig. (3). D’Orazio et al. employed a
novel chiral nano-LC-MS for analysis amino acids in orange juice profiling. The
complete set-up used for nano-LC-MS analysis of amino acid enantiomers is
shown in Fig. (4). Luo et al. adopted chiral liquid chromatography and tandem
mass spectrometry for simultaneous analysis of bambuterol and its active
metabolite terbutaline enantiomers in rat plasma, the typical chromatograms are
shown in Fig. (5).
4.2. LC-CD
It is always required to determine the enantiomeric purity in the quality control of
chiral drugs production. At present, the importance of the circular dichroism (CD)
spectroscopy is increasing importance in pharmaceutical analysis because of the
continuous appearance of commercially available CD detector in liquid
chromatography Fig. (6) shows the basic components of the CD detector.
HPLC with CD detector analysis (LC-CD) can establish a correlation between the
absolute configuration of separated enantiomers and their characteristic CD
transitions. So, LC-CD hyphenated technique is playing a gradual key role for
determination of absolute configuration of chiral drug, especially in drug
discovery. There are lasting LC-CD applications reported about determination of
absolute configuration for enantiomers. Iwasa et al.employed on-line HPLC-CD in
combination with HPLC-MS for analysis of benzylisoquinoline alkaloids,the
chromatogram is presented in Fig. (7). A novel chiral detector, a circular-dichroism
thermal lens microscope (CD-TLM), was developed to realize sensitive and
selective detection of small volume chiral samples on a microchip (2006) [135].
Thermal lens spectrometry (TLS) is a kind of photothermal spectrometry which
holds promise for overcoming the low sensitivity of absorption-based detection
methods, and the principle of CD-TLM is illustrated in Fig. (8). This novel chiral
detector is more than 250 times higher sensitivity versus that in a CD
spectrophotometer for pure enantiomer detection. Ranjbar et al. (2009) [136]
introduced circular dichroism techniques for biomolecular and nanostructural
analyses. The result indicated circular dichroism is a powerful technique for
evaluating the conformation of polypeptides and proteins. In a word, the
development tendency of CD detector is aiming to improve its sensitivity to detect
smaller amounts of enantiomers
4.3. LC-OR
Some applications for OR detectors have received the most attention to date. The
most fundamental is to verify the optical activity of the compounds of interest and
to assign either a positive or negative rotation of the structure. LC-OR can also be
adopted for the identification and characterization of impurities. However, this
application has received little attention to date, primarily due to the relatively low
sensitivity of most OR detectors and the need for optically pure standards to
facilitate identification. Kott et al. made a meaningful evaluation of four
commercial HPLC chiral detectors (three polarimeters and a circular dichroism
detector). Moreover, LC-OR hyphenated technique has been developed for
determining the change of enantiomeric excess in the chiral synthesis in order to
confirm the reaction termination.

5. SOME NEW DEVELOPMENTS AND APPLICATIONS IN HPLC

CHIRAL SEPARATIONS
Today, rapid analysis for chiral pharmaceuticals has become an inevitable trend.
Fmoc- and Z-derivatives of natural and unnatural sulfur containing amino acids
were separated by micro-HPLC with very short time. The separation was carried
out in microbore columns packed with a new material based on Ristocetin A
bonded to 3.5 μm silica gel. The major advantage of micro-HPLC is much less
consumption of expensive packing material, solvent and sample. Furthermore, the
small particle size leads to higher efficiency and ideal resolution with shorter
retention times.
Currently, particle size can be decreased even more, and the use of columns packed
with sub-2 μm particles working under ultra-high pressure have been developed
over the last 5 year. So, the use of columns packed with sub-2 μm particles in
liquid chromatography with very high pressure conditions (known as Ultra High
Pressure Liquid Chromatography, UHPLC) was investigated for the fast
enantioseparation of drugs. Guillarme et al.employed two alternative approaches
for evaluation for rapid chiral analysis by UHPLC using amphetamine derivatives
and β-blockers as model compounds. Using this strategy, it was possible to achieve
the robust separation presented in Fig. (9). As a result 10 enantiomers from 5
different β-blockers were well separated in less than 3 min, which demonstrated
that the developed method could be applied for the establishment of optical purity
of pure compounds at low levels in very short analysis times

CSPs associated with simulated moving bed (SMB) technology have also shown
excellent performance in the separation of racemates. This technology has been
mainly used in the area of chiral purification, where the resolution of enantiomers
is usually a binary separation task with low selectivity and high operational cost.
Therefore, SMB is not only the method to prepare few grams of the chiral drug
required by preliminary clinical tests but even more so most suitable way for the
large-scale production. A laboratory-scale SMB unit made in home was adopted in
an eight-column configuration (two column per section), as shown in Fig. (10).
Rajendran et al. reviewed simulated moving bed chromatography using for the
separation of enantiomers. After realizing its potential for enantiomer separations,
now the SMB technology is an important component of the separation toolbox in
the pharmaceutical industry, particularly to manufacture single pure enantiomers of
racemic drugs. Nowadays, multidimensional high performance liquid
chromatography technique is a powerful tool for analysis of complicated samples
especially biological fluids. It is a powerful tool for chiral analysis too, which can
be used to determine not only drugs’ metabolites in plasma but also value of
enantiomeric excess (e.e.%). Mullett et al. provided an overview of the existing
literatures with emphasis on advances in automated sample preparation methods
for liquid chromatography. The column configurations including multi-
dimensional-column mode can provide additional dimensions of selectivity.
Albendazole metabolites were analyzed by direct injection of bovine plasma on
multidimensional high performance liquid chromatography , and the column-
switching system employed is illustrated in Fig. (11). Shi et al.reviewed a recently
developed technique that couples HPLC with on-line, post-column (bio)chemical
assays and parallel chemical analysis to screen and identify bioactive compounds
from complex mixtures without the need for cumbersome purification and
subsequent screening. Those strategies have proved to be very useful for rapid
profiling and identification of individual active components in mixtures, hence to
provide a powerful method for natural product-based drug discovery. This system
coupled with chiral columns can be employed for analysis of complicated samples
with the high selectivity and sensitivity of detection. The on-line assays can always
be adopted in a qualitative way and known active compounds can be screened and
identified rapidly, while novel active compounds could be further investigated after
target purification.
6. MECHANISM OF CHIRAL RECOGNITION
Although there are so many commercially available chiral columns used for chiral
analysis, it is difficult to select suitable CSPs for efficient resolution of specific
enantiomers rapidly. Therefore, it is very important for us to understand the
mechanism of enantioseparation. Today, the intermolecular interactions between
chiral selector and enantiomers can be calculated with quantum mechanics,
molecular mechanics and molecular dynamics. The various molecular modelings
have been established by X-ray crystallography, NMR and computer simulation in
order to study chiral discrimination mechanism. Among them, molecular modeling
is considered as a practical tool for evaluating the complex interactions taking
place as analytes migrate through a chromatographic column, which could provide
detailed information at the atomic level about how chromatographic process
undergoes. Several molecular simulation theories have been employed to simulate
the liquid chromatography process in enantioseparation. Chemometric analysis of
enantiospecific retention data was first described by Francotte and co-workers.
These authors applied multiple regression analysis to relate enantioselective
separation on cellulose triacetate and cellulose tribenzoate columns to structural
descriptors of analytes. In order to study the retention and chiral recognition
mechanism, the method of quantitative structure-enantioselectivity retention
relationships (QSERR) was investigated from the quantitative equations
established between the chromatographic retention of enantiomers and their
molecular descriptors of physico chemical properties. QSERR was first introduced
as a special term in a report on studies of HPLC data determined on human serum
albumin (HSA) column. Then, QSERR has been extensively studied since decades.
Heberget reviewed Quantitative structure (chromatographic) retention relationships
(including QSERR) from 1996 to 2006. QSERR models can be used for successful
classification of chiral drugs of various compound classes and/or chromatographic
columns (systems), which is very useful for HPLC practitioners. Consequently, in
QSERR studies one can address either variation in the non-enantiospecific strength
of solute-CSP interactions due to differences in the chemical structure and
properties (such as polarizability, number of hydrogen atoms, etc.) which increases
the overall retention of both isomers or the forces which lead to chiral
discrimination. Moreover, Lipkowitz introduced the atomistic modeling of
enantioselection in chromatography, and the mechanism of chiral recognition for
five types of CSPs has been studied and direct interactions are calculated between
CSPs and analytes. The chromatographic process can be well analyzed using
molecular dynamic model theories. The determination of the adsorption sojourn
times and the number of adsorption–desorption or mass-transfer events can give a
practical view of the molecular process of separation. The studies of various kinds
of molecular modeling for chiralseparation by LC have been investigated
extensively. With researching the chiral recognition mechanisms at the molecular
level, we can not only explain the experiment phenomena but also predict the
results. It can guide us to adjust chromatographic conditions in order to improve
the resolution of enantioseparation. What is more important, the new types of CSPs
can be designed to realize the expected separation.
7. CONCLUSION
A major trend in LC for the separation of drug enantiomers is clearly heading
toward faster and more efficient separation with comparable or improved
separation capability. The development of liquid chromatographic procedures
focuses on usage of efficient novel materials in column chromatography and
hyphenated techniques with highly selectivity. After the rapid development in
many years, enantioselective liquid chromatography, particularly HPLC on CSPs,
has become an indispensable part of drug discovery not only in daily chiral
analysis but also in the fast preparation of drug molecules. As more chiral selectors
are being developed and commercialized as CSPs, a systematic and deeply
understanding of the chiral recognition mechanisms at the molecular level is
essential for the future, so that researchers can rapidly select or even create suitable
CSPs for efficient resolution of specific enantiomers in one day. Moreover, by
using hyphenated techniques (LC-MS, LC-CD, LC-OR), we can know more
information about molecule structure, pharmacokinetics, spatial configuration,
eluting order for drug enantiomers. With the advances and promotion of related
techniques, recent developments in HPLC enantiomeric separation, including
micro-columns, small particle columns, simulated bed moving chromatography
and multidimensional chiral high performance liquid chromatography, have been
reviewed in this paper. Collectively, these advances will lead to persistent
improvement of chiral separation capability.
ABBREVIATIONS
API = Atmospheric pressure ionization
APPI = Aatmospheric pressure photoionization
AVI = Avidin
AGP = α1-acid glycoprotein
BSA = Bovine serum albumin
CBH I = Cellobiohydrolase I
CD = Circular dichroism
CD = Cyclodextrin
CDR = Chiral derivatization reagents
CD-TLM = Circular-dichroism thermal lens microscope
CMPA = Chiral mobile phase additives
CSPs = Chiral stationary phases
HAS = Human serum albumin
HPLC = High performance liquid chromatography
ILs = Ionic liquids
MIP = Molecularly imprinted polymers
MS = Mass spectra
OMCHI = Ovomucoid from chicken egg whites
OR = Optical rotation
SMB = Simulated moving bed
TLS = Thermal lens spectrometry
UHPLC= Ultra high performance liquid chromatography
Separation of Enantiomeric Compounds Using Chiral HPLC
Systems. A Brief Review of General Principles, Advances, and
Development Trends*
Introduction
A remarkably rapid growth of the field of direct chroma- tographic resolution of
enantiomeric species, which prob- ably started about 20 years ago in GC with the
introduction of highly efficient capillary columns containing chiral acylamino
ester-type stationary phases by GiI-Av et al. [1] and, in LC, with the introduction
of highly selective chiral ligand-exchange-type phases by Davankov and Rogozhin
[2-3], resulted in a whole series of important theoretical and practical
achievements. In addition to Pasteur's classical principles of resolving racemic
mixtures into constituent enantiomers, new stereochemical concepts have been
formulated and proven experimentally. Some understanding of the mechanisms of
intimate intermolecular inter- actions between solute enantiomers and functional
groups of the stationary phase in many chromatographic systems, e.g. ligand-
exchanging and charge-transfer-complexing ones, has been gained. Experimental
possibilities in biochemical and pharmaceutical research for the determination of
the enantiomeric purity of chiral compounds, for the preparation of thousands of
new chiral xenobiotics of an un- precedented purity and for the study of metabolic
path- ways of chiral drugs in living organisms have been dramatically increased.
Due to these advances the number of re- search groups dealing with optically
active compounds has multiplied, resulting in a real information explosion at the
borders of chromatography with stereochemistry, pharmacology, biochemistry.
Dozens of reviews (e.g. Ref. [4-18]) discuss numerous successful resolutions of
enantiomeric compounds in di- verse chiral HPLC systems. Therefore, in the
present review it is more appropriate to concentrate on the general stereo-chemical
aspects of functioning within chiral HPLC systems.
General Principles of Chiral Chromatographic Separations
1. Thermodynamics of Chiral Resolution
Enantiomeric substances, though consisting of non-super- imposable molecular
species, display identical* physical and chemical properties both in the gaseous
and condensed states as well as in solution. As a basic principle only chiral
molecular structures (or chiral irradiation) can distinguish between two
enantiomers. Thus, enantiomeric resolutions are only feasible in chromatographic
systems that contain an appropriate chiral selector.
The chiral selector can either be incorporated in the stationary phase - via covalent
bonds to the sorbent matrix ("Chiral Stationary Phase", CSP) or permanent
adsorption onto the sorbent surface ("Chiral Coated Phase", CCP) - or it can be
added to the eluent ("Chiral Mobile Phase", CMP).
The difference in the interaction of the chiral selector with the two enantiomers
under resolution is called enantio- selectivity [2, 20]. In chromatography we only
deal with thermodynamic enantioselectivity effects, i.e. formation of labile
diastereomeric adducts ABR and ABs of the chiral selector A with the enantiomers
B R and B s of the solute B, the adducts differing in their stability (in the case of
CSP and CCP) and/or in their interphase distribution ratio (in the case of CMP).
This difference in stability constants, KR and K s , of the two diastereomeric
adducts relates to the difference in their free energy, ~G, and the column
enantioselectivity, o~, through a simple exponential expression
ΔΔG =-RTIn(KR/Ks)≈-RT In α
A minor thermodynamic enantioselectivity of ΔΔG = 0.024 kJ/mol, corresponding
to an e value of 1.01, would already suffice for many enantiomeric pairs to be
resolved using modern chromatography techniques. With rising ΔΔG values, the
column selectivity rises exponentially. If one could double ΔΔG one would
increase e to the square of its initial value. Pirkle and Pochapsky [21] demonstrated
generation of extreme selectivity in chiral recognition on the basis of the above
considerations. On a chiral stationary phase of the following structure

the authors observed resolution of N-(3,5-dinitrobenzoyl)- leucine n-hexylamide

with a selectivity value of e = 10.5 (in 30% 2-propanol in hexane). This selectivity
corresponded to stronger binding of the S-solute by a value of ΔΔG = 5.93 kJ/mol,
When a bis-solute was prepared that contained two asymmetric centers

situated at a distance that would allow simultaneous inter- action of the bis-solute
with two chiral sorbtion sites of the bonded phase, the value of ΔΔG was doubled.
Indeed, selectivity of the column with respect to the SS- and RR- enantiomers of
the bis-solute was observed to rise to the square of the initial value and reach the
record value of = 121.
As a thermodynamic quantity, the stability constant, K, of each of the two
diastereomeric sorption complexes should be temperature dependent: - RT In K =
ΔG = ΔH-TAS. This leads [22, 23] to a linear dependence ofthe enantioselectivity
ΔΔG and Ine on the inverse of temperature l/T, It is important to note, that the
resolution selectivity may both fall or rise with rising temperature, depending on
the position of the characteristic temperature, Tiny., at which the stability constants
of the two diastereomeric sorbtion complexes appear equal and, consequently, the
resolution vanishes. On passing this temperature the sign of the enantioselectivity
effect should reverse [23]. Such an inversion of elution order of two enantiomers
has been observed in GC experiments by Watanabe et al. [24] and more recently
by Koppenhoefer et al. [25] and Schurig et al. [26, 27]. (The latter authors discuss
the competition of two complexation mechanisms as an alternative to the above
thermodynamic explanation of the enantioselectivity inversion). In liquid
chromatography, reversal of the elution order of
t I t . . 2,4,2,4.tetrahydroxy-6,6-dlmethyl-blphenyl enantiomers from a starch
column on raising the temperature from 20 to 59~ was reported [28].
Usually, the temperature range accessible in LC is too small to allow the
enantioselectivity inversion temperature to be located experimentally. As a rule,
the selectivity falls with increasing temperature, which is especially remarkable in
the case of the crown ether-incorporating CSP "Crownpack CR". There is also one
example described, namely, the resolution of racemic N-benzylproline on the
Cu(ll) form of a L-proline-containing polystyrene-type ligand exchanger, where
higher column temperatures resulted in increased e-values [29]. It was shown [30]
that the enantioselectivity of bis-(N-benzyl prolinato)copper formation is governed
by the entropic contribution abnormally dominating over the enthalpic one.

Chiral Drug Separation

What are Chiral Molecules?
A molecule with an asymmetric carbon center has a unique three-dimensional
shape and is called chiral, from Greek cheir meaning ‘‘hand.’’ A chiral molecule
and its mirror image are not completely identical: they differ in their
‘‘handedness’’ and are not superimposable. Such chiral molecules are called
‘‘enantiomers,’’ from Greek enantios, meaning ‘‘opposite.’’ A human right hand
and left hand are enantiomers: they are mirror images, but a ‘‘left’’ glove cannot be
worn on a right hand. The arrangement of thumb and fingers in three dimensions
makes a right hand and a left hand distinctly different from each other. The two
different forms of a chiral pair comprise the same number and types of atoms, and
they are commonly described as D- andL-isomers with reference to their ability to
rotate polarized light. An equimolar mixture of enantiomers is called a
‘‘racemate.’’ Fig. 1 depicts 2-butanol. The top two structures are mirror images.
Below, ‘‘right-handed’’ 2-butanol has been rotated so that the OH group points to
the left for comparison with ‘‘left-handed’’ 2-butanol. Although the carbon
frameworks of the two molecules align, the position of the hydroxyl group is
different. In the rotated version of right-handed 2-butanol, in the lower left panel,
the hydroxyl group points into the page; in left-handed 2-butanol, in the lower right
panel, the hydroxyl group points out from the page. It is impossible to align the
two molecules completely without breaking bonds. Left-handed and right-handed
forms of a molecule can have profoundly different properties in a biological
context. In the food industry, for instance, left-handed limonene smells like
lemons, while the right-handed molecule smells like oranges. Different
enantiomers can differ widely in their biological properties because chirality is
related to the three-dimensional structure, and one form may be more suitable for
specific interaction with a biological molecule, such as a receptor, enzyme, etc.
Chiral Drugs
Most synthetic drugs developed in the past were not chiral, though some were.
Drugs developed from natural products are largely chiral. Currently, about 40% of
the drugs in use are known to be chiral. A report from Technology Catalysts
International, Falls Church, VA, states that worldwide sales of chiral drugs in
single-enantiomer dosage forms grew at an annual rate of more than 13% to $133
billion in 2000, and that sales could hit $200 billion by 2008.[1] About one-third of
all dosage-form drug sales in 2000 were single enantiomers. By geography, the
United States is the largest consumer of enantiomeric fine chemicals, contributing
60% of the worldwide total. Drug companies continue develop chiral drugs as
single enantiomers and use chirality to manage drug life cycles. Most
commercially available drugs are both synthetic and chiral. A large number,
however, are still marketed as racemic mixtures. Only about one-third of drugs are
administered as pure enantiomers. The respiratory drug montelukast (Merck), the
antirheumatoidal infliximab (Centocor), the ophthalmic drug for the treatment of
glaucoma latanaprost (Pharmacia), and the prostate hyperplasia agent tamsulosin
(Boehringer Ingelheim Pharmacy) are among the best-selling single-enantiomer
drugs. The chiral drug (S)-(þ)-ibuprofen is marketed as fast-acting, and it reaches
therapeutic concentrations in blood in 12 vs. 30min for racemic mixtures. Allegra,
an isomer from a metabolite of seldane, is used as anallergy medication. Table 1
lists further examples of chiral drugs and their bioactivities. The enantiomeric
forms of a drug can differ markedly in potency, toxicity, and behavior in biological
systems.
Importance of Chiral Drug Separation
In the early 1980s analytical chiral separation was a rather difficult task, and
preparative synthetic and separation methods were not as advanced as today.
Nevertheless, it was clear that chiral drugs should be enantioseparated and that
each enantiomer should be used separately. Nowadays, enantiomers are considered
distinctly different compounds, as enantiomers of drug substances may have
distinct biological interactions and, consequently, profoundly different
pharmacological, pharmacokinetic, or toxicological activities.[2] The body is
highly chiral selective; it will interact with each racemic drug differently and
metabolize each enantiomer by a separate pathway to produce different
pharmacological activity. One isomer may thus produce the desired therapeutic
activities, while the other may be inactive or produce unwanted side effects (see
Table 1). Even when side effects are not serious, the inactive enantiomer must be
metabolized and thus represents an unnecessary burden for the organism. One
chiral form of the drug naproxen has 28 times the anti-inflammatory activity of the
chiral relative. One isomer of dopamine, used to treat Parkinson’s disease, acts on
nerve cells to control tremor, while the other is toxic to nerve cells. Racemic
mixtures of the drug thalidomide were marketed to pregnant women in the 1960s
to counter morning sickness. Therapeutic activity, however, comes exclusively
from the (R)-(þ)enantiomer. It was discovered only after several hundred birth
defects had resulted from administration of thalidomide that the (S)-(þ)-enantiomer
is teratogenic. By then, consideration of chirality has become an integral part of
drug research and development and of the regulatory process. In general, use of the
more active isomer of a drug has the following advantages:
 Fewer or diminished side effects, which may result from the unwanted
isomeric form.
 Automatically halved dosage for a patient.
 Decreased waste due to decrease in manufacturing of unwanted isomer.
 New commercial opportunities for ‘‘racemic switching:’’ a drug previously
marketed as a racemate can be redeveloped and introduced as an
enantiomerically pure form, possibly useful for extending patent protection
of a key product.
Table 1 Examples of chiral drugs and functions
Chiral drugs Bioactivity
AlbuterolD-isomer may provoke airwayconstriction;L-isomeravoids side effects
Ethambutol The (S,S)-form of ethambutol is a tuberculostatic
agent; the (R,R)-form causes optical neuritis that
can lead to blindness
Levodopa The levodopa (L-dopa) is a Parkinson’s disease
agent; the D-form causes serious side effects,
such as granulocytopenia
PenicillamineThe (S)-enantiomer has antiarthritic activity;
the (R)-form is extremely toxic
PropanololRacemic compound is used as drug; however,
only the (S)-()-isomer has the desired b-
adrenergic blocking activity Propoxyphene a-
L-isomer is antitussive (cough); a-D-isomer is
analgesic (pain)
Thalidomide The (S)-isomer has the desired antinausea
effects; the (R)-form is teratogenic and causes
fetal abnormalities, such as severely
underdeveloped limbs

There are obvious benefits to studying the properties of the enantiomers of a chiral
drug molecule with respect to therapeutic efficacy and safety. In view of this, since
1992 the FDA and the European Committee for Proprietary Medicinal Products
have required that the properties of each enantiomer be studied separately before
decisions are taken to market the drug as one of the enantiomers or as a racemate.
[3] This requires powerful means of chiral drug detection and separation. In
addition, there is increasing awareness of the need to reevaluate the properties of
individual enantiomers of currently marketed racemic drug molecules. The effect
has been a significant increase in demand for sensitive chiral analytical and
separation methods. At the same time, the number of new chemicals entering
development within the pharmaceutical industry has increased significantly. By
now most drug companies have clear guidelines recommending that the
enantiomers of all chiral bioactive molecules be separated and tested. The ideal
way to obtain pure drug enantiomers would be enantioselective synthesis. This,
however, is rarely practical, usually complicated, and almost always expensive.
There is little control over which chiral form of a chemical compound will be
formed during a typical production process. This lack of control generally results
in production of equal amounts of the various possible chiral forms of a compound.
Often, therefore, separation of intermediates or final products from a racemic
mixture is required. Increasing interest is being paid to development of methods of
efficient, high throughput, and sensitive chiral separations, control of chemical
synthesis, assessmentof enantiomeric purity, and determination of
pharmacodynamics. The various examples of different therapeutic, toxicological,
and pharmacokinetic properties of the enantiomers of chiral drugs provide a strong
impulse for the development of techniques for chiral drug separation. Enantiomers
can differ in absorption, distribution, protein binding, and affinity to the receptor.
Such properties have been exploited for the development of powerful techniques
for achieving analytical-scale chiral separations, quantifying minor enantiomeric
impurities in chiral drugs, and preparing gram to multi-ton amounts of
enantiomerically pure chiral drugs. Chromatographic techniques, such as HPLC,
GC, thin layer chromatography, and supercritical fluid chromatography have been
developed for chiral separations. Capillary zone electrophoresis, capillary gel
electrophoresis, electrokinetic chromatography, and capillary
electrochromatography have proved powerful alternatives to chromatographic
techniques.
CHIRAL DRUG SEPARATION PRINCIPLES AND TECHNIQUES
Principles of Chiral Separation and Chiral Selectors
Principles of chiral separation
Separation of enantiomers has been achieved using GC, HPLC, and CE. Chiral
recognition generallydepends on a minimum of three simultaneous interactions
between the selector and selected—the so-called three-point-interaction rule of
Dalgliesh (Fig. 2).[4,5] At least one of these interactions must be stereoselective to
form diastereomeric complexes, and thereby enable chiral separation. The principle
task of chiral separation is to create the selectivity essential for separation of
stereoselectively different forms of compounds, which may be recognized as such
only during the interaction with a chiral selector. This is the separation principle
for chromatographic techniques and also for chiral CE. In chromatographic chiral
separation, there is a distribution of analyte between two immiscible phases, and
these should exhibit different mobilities. Commonly one phase is mobile and the
other is stationary. In chiral CE there are not two immiscible phases present but
pseudophases at best, or even only one monophasic, homogenous system. Chiral
recognition, however, occurs at the molecular level, not on the macroscopic level
of the phases. A separation technique therefore must allow the transfer of the
molecular level event (in this case chiral recognition) to macroscopic phenomena:
different retention times of enantiomers in chromatography and different effective
mobilities of enantiomers in electrophoretic techniques. Immiscibility of phases is
a prerequisite in chromatographic separation because pressure as a driving force
cannot select a given component from several species in the same phase. Under
certain circumstances, however, electrically driven mobility can be selective for
one or several species residing in the same phase. So, the immiscibility
requirement of the two phases does not apply to chiral CE. In other words,the
principal difference between chromatographic techniques and CE is that pressure
cannot distinguish between different molecular components in a monophasic
system, whereas electrically driven mobility can do this under certain
circumstances. The phenomenon responsible for chiral separation is the same in
chromatographic and electrophoretic techniques: enantioselective interaction
between the analyte enantiomers and a chiral selector.

Chiral selectors
Enantiomers are distinguished on the basis of their interaction with a chiral
selector. Development of chiral selectors or chiral stationary phases (CSPs) for
GC, HPLC, and CE has rapidly opened a new dimension in the area of chiral drug
separation techniques. There are different chiral selectors available for
enantiomeric separation of drugs and pharmaceuticals. Finding a suitable chiral
selector, whether immobilized on a solid support (GC, HPLC) or added to a
running buffer (HPLC, CE), is still often based on trial and error. A few
predictions can be made, however, if common structural elements are present.
After a selector has been chosen, variables, such as the nature, ionic strength, and
pH of buffer, can be varied, as can presence of organic modifiers, temperature, and
so on. Among available selectors, native and derivatized cyclodextrins (CDs) are
the most widely used ones at this time. A majority of drug and pharmaceutical
applications have been achieved with CDs. Use of CDs as chiral selectors is the
subject of a number of reviews (e.g., Refs.[6,7].). Native CDs are cyclic
oligosaccharides consisting of six a-CD-, seven b-CD- or eight g-CD-
glucopyranose units. A truncated cone provides a hydrophobic cavity; the exterior
surface, surrounded by hydroxyl group, is hydrophilic. Low UV absorbance, low
cost, and water solubility are attractive properties of CDs for use as chiral
selectors. In addition to CDs, other chiral selectors, such as natural and synthetic
chiral surfactants, crown ethers, proteins, oligo- and polysaccharides, macrocyclic
antibiotics, and chiral ligands have been applied to chiral separations. Important
selectors and some chiral recognition mechanisms are given in Table 2. Some
chiral selectors developed thus far can efficiently resolve enantiomers of various
important drugs.
Chiral Drug Separation Techniques
The main methods used for chiral drug separation are GC, HPLC, and CE.[2,4,6–
25] Other techniques, such as chiral crystallization and enzyme-based kinetic
separation, have also attracted attention.[26]
Applications of GC to chiral separation
The first separation of enantiomers was achieved by Gil-Av, Feibush, and Charles-
Sigler[9] using capillary GC. Separation of enantiomers using CSPs involves
hydrogen bonding, coordination, and inclusion. Typical chiral selectors include
modified CDs, derivatized amino acids, and terpene-derived metal coordination
compounds. The scope and limitations, applications, and mechanistic
considerations of chiral separation by GC have been reviewed by Schurig and
Francotte.[10,11] The main applications of enantiomeric separation by GC concern
precise determination of enantiomeric composition of chiral research chemicals,
drugs, intermediates, metabolites, pesticides, flavors and fragrances, etc.
CHIRBASE, a database of chiral compounds, provides comprehensive structural,
experimental, and bibliographic information on successful and unsuccessful chiral
separations, and rule sets for each CSP and information about the processes of
chiral separations.[27] According to CHIRBASE, an appropriate CSP is available
for almost every racemic mixture of compounds ranging formapolar to polar. Some
22,000 separations of enantiomers, involving 5,500 basic chiral compounds and
documented in 2,200 publications, have been achieved by GC. This method is
particularly suitable for volatile compounds such as inhalation anesthetic agents,
e.g., enflurane, isoflurane, desflurane, and racemic a-ionone. A particularly
attractive feature of GC, one that distinguishes it from liquid chromatography
methods, is the lack of a sensitive dependence on solvents, modifiers, and gradient
elution systems. Prerequisites for the use of GC, however, are volatility,
thermostability, and resolvability of the chiral analyte. Obviously, this restricts the
utility of the method.
Applications of HPLC to chiral separation
Chromatographic methods have dominated separation of enantiomers during the
past several decades, especially HPLC.[4,15–17] Numerous book chapters and
review articles deal with the separation of chiral drugs by this method (e.g., Refs.
[2,6,12–14].). Chiral HPLC is more versatile than chiral GC for enantiomeric
separation because it can separate a wide variety of nonvolatile compounds. It can
be used to develop fast and accurate methods of chiral drug separation, and it
allows on-line detection and quantitation of both mass and optical rotation of
enantiomers when appropriate detection devices are used. Current chiral separation
methods using liquid chromatographic techniques can be divided into two
categories: a direct method based on diastereomer formation on CSPs or in mobile
phases, and an indirectmethod based on diastereomer formation by reaction with a
homochiral reagent. Direct chiral separation using CSPs is more widely used and
predictable in mechanistic terms than methods involving chiral additives in the
mobile phase. To date, more than a hundred HPLC CSPs are commercially
available. No single CSP can be considered universal; none has the ability to
separate all classes of racemic compounds. Three components should be
considered in developing a chiral separation method: analyte, CSP, and mobile
phase. The key to successful chiral separation of a particular class of racemates on
a given CSP is awareness of possible chiral recognition mechanisms. The
enormous increase in recent years in number of groups working on chiral
chromatography has led to a rapid and impressive accumulation of data in
CHIRBASE.[27] Some examples of chiral HPLC separations of racemic drugs are
the following. Typical chromatograms of the simultaneous determination of
isopyramide and its active metabolite, mono-N-dealkyldisopyramide, in drug-free
human plasma, human plasma spiked with disopyramide and mono-N-
dealkyldisopyramide, and treated subject plasma are presented in Fig. 3.[4] Chiral
HPLC has been used to separate chlorpheniramine and its main monodesmethyl
metabolite, verapamil and its metabolite, and tramadol and its metabolite.[15–17]
Applications of CE to chiral separation
CE has become widely popular for enantiomer separation over the past decade as a
very powerful complementary or alternative technique to HPLC in pharmaceutical
science and industry. Several chiral separation principles successfully applied in
HPLC have been transferred to CE. The first chiral separation by CE was by
Gassmann, Kuo, and Zare in 1985.[18] This approach offers key advantages such
as high efficiency, feasibility of incorporating a large number of chiral selectors
that greatly facilitates method development, small amounts of chiral selector and
solvents, speed of analysis, low overall cost, and minimal environmental impact.
The use of low-UV wavelength (e.g., 200nm) in CE allows detection of impurities
with poor chromophores, which may be difficult or impossible to detect by other
methods. CE is suitable for charged and polar compounds for which
chromatographic methods are not very strong. Several comprehensive review
articles have appeared in recent years dealing with general aspects and applications
of chiral CE separation.[8,19–21] A comprehensive list of more than 280 drugs
separate by chiral CE, including the respective chiral selectors and background
electrolytes, appears in a review article by Gu ¨bitz and Schmid.[21] Chiral CE has
alsobeen the theme of several special issues of Electrophoresis and Journal of
Chromatography A. Chiral separation in all CE techniques, including capillary
zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and capillary
isoelectric focusing (CIEF), relies on enatioselective noncovalent= intermolecular
interactions between the analyte and a chiral selector, which may be expressed as
effective mobility difference (CZE and CGE), stereoselective shift of the acid–base
equilibrium (CIEF), etc. Although the fundamental enantioselection mechanisms in
CE are the same as in chromatography, migration is driven by electrophoresis.
Enantiomers of a chiral drug compound will have the same charge density, so
chiral separation in CE is not commonly based on electrophoretic separation, in
which different migration velocities are an effect of different charge densities of
analyte components. For enantiomers, both the electroosmotic flow and the
electrophoretic mobility of the analyte are equally nonstereoselective. What
distinguishes enantiomers in chiral CE is their interaction with a chiral selector.
Chiral separation by CE can be achieved by a direct or an indirect method. Direct
separation is the more common approach. The chiral selector is dissolved in the
running buffer, where it interacts selectively with the enantiomers to form
reversible and transient diastereoisomeric or inclusion complexes of differing
effective mobility. In indirect chiral CE separation, the enantiomers form covalent
diastereomeric derivatives with a chiral reagent.
Fig. 3 Chromatograms showing analysis of disopyramide and mono-N-
dealkyldisopyramide enantiomers in plasma: (A) blank plasma, (B) plasma spiked
with 625ng=ml of disopyramide and mono-N-dealkyldisopyramide enantiomers,
(C) plasma sample from a healthy volunteer collected 6hr after administration of
100mg of Dicorantil. Peak assignments: 1, (S)-(þ)disopyramide; 2, (R)-()-
disopyramide; 3, 4, metoprolol; 5, (S)-(þ)-mono-N-dealkyldisopyramide; and 6,
(R)-()mono-N-dealkyldisopyramide. Chromatographic conditions: Chiralpak AD
column (2504.6mm I.D., 10mm particle size); hexane–ethanol (91:9, v=v) mobile
phase plus 0.1% diethylamine; 1.2ml=min flow rate; and detection at 270nm.
(From Ref.[4].)

A chiral selector is unnecessaryin this approach because of the different

electrophoretic mobilities of the diastereomeric derivatives. Examples of chiral CE
separations of racemic drugs are the following. (R)-()-ketoprofen has successfully
been separated from ketoprofen and detected (Fig. 4).[25] (S)-(þ)-ketoprofen is the
active component. Also, simultaneous chiral separation of a basic drug compound,
2(R)-N-[1-(6-amino-pyridin-2-ylmethyl) piperidin-4-yl]-2-[(1R)-3,3-
difluorocyclopentyl]-2-hydroxy-2phenyl-acetamide, and its chiral acidic
intermediate, (R,R)1-(2,2-difluorocyclopentyl)-phenylacetic acid, has been
achieved by CE using a single-isomer CD, octakis (2,3-diacetyl-6-sulfo)-g-CD
(ODAS-g-CD).[22] Carnitine has been separated using 50mM DM-b-CD in 20mM
phosphate buffer (pH 4.3) as chiral selector.[23] The separations are done at 30C in
a fused-silica capillary, dynamically coated with triethanolamine present in the
background electrolytes.
Similarities and differences in chiral separation by chromatographic and capillary
electrophoretic techniques
HPLC remains the dominant technique for chiral separation in industry. CE has
become well accepted in academic laboratories. Current GC, HPLC, and CE
instruments are automated. Chiral separation in CE relies on a chromatographic
separation principle. Nevertheless, there are significant differences, as shown in
Table 3, between these techniques. The property ofelectrophoretic mobility in
chiral CE in particular, and its ability to be selective for the analytes residing in the
same phase, is responsible for all the differences. Another important point is that in
chromatographic techniques, except with a chiral mobile phase additive, the
analyte is virtually immobile when associated with a chiral selector. By contrast, in
CE the analyte selector complex is commonly mobile. In studies of a chiral drug
candidate and its possible metabolites in preclinical and clinical Phase I to Phase
III, most of the biological sample matrices, such as urine, plasma, serum, saliva,
cerebrospinal fluid, and tissue homogenates, are more compatible with CE than
chromatographic techniques. Moreover, it is not possible in GC and difficult in
chiral HPLC to achieve the chiral separation of a drug and its phase I and phase II
metabolites in a single run. By contrast, this is possible with chiral CE, as in the
simultaneous chiral separation of Phase I and Phase II metabolites of chiral
antihistaminic drug dimethindene.[24] In summary, there are significant
differences between chiral separations in pressure-driven HPLC and electrically
driven CE systems. These differences are advantageous in that they make the
techniques complementary. The rules and dependencies observed in one technique,
however, are not necessarily applicable to the other.Recently developed techniques
for chiral separation: chiral membranes
HPLC is useful because of its preparative-scale capability. The method is generally
slow and laborintensive, however, requiring specialized engineering approaches
for acceptable throughout. In comparison, chiral membrane separation offers
significant advantages in simplicity, cost, and throughput.[28] Polypeptides or
modified polypeptides have been tested for use in chiral membrane separation. The
partition behavior of optical isomers will largely be influenced by the structure and
number of ‘‘recognition sites’’ in the polypeptide membrane. Polypeptides could
be designed on demand and used to build polypeptide films=membranes.[29]
Polypeptide membranes have shown very high enantiomer permeation rates with
encouraging selectivity for chiral drug separation.[30] Peptide hydrophobicity,
molecular weight, and secondary-structure formation propensity of specially
designed peptides will be considered in future work on chiral separation
membranes, as polypeptides could be designed to behave like certain proteins in
human body and thus to achieve high biomimetic enantioselectivity. This approach
would appear to promise very high enantioselectivity and high permeation rates.
CONCLUSIONS
It is necessary to consider the chiral nature of a compound in drug research and
development and the drug regulatory process. Enantiomers of all chiral bioactive
molecules must be separated and tested. The FDA and regulatory authorities in
Europe, China, and Japan have provided guidelines indicating that only the active
enantiomer of a chiral drug should be brought to market. Chiral techniques have
been developed for the separation and analysis of chiral drugs. Among these,
HPLC based on CSPs is widely employed for the assays of drug enantiomers in
pharmaceutical preparations and biological fluids. More recently, CE plus chiral
selector additives to the running buffer have been used for the same purpose. In
both chromatographic and electrophoretic methods, different types of chiral
selectors, including CDs, crown ethers, quinine, chiral surfactants,
polysaccharides, proteins, and macrocyclic antibiotics, have successfully been used
for chiral separation of drugs and drug candidates.In many cases, HPLC and CE
are complementary with respect to enantioselectivity. The development of novel
separation techniques is an active area of research. Ones showing high throughput
and high enantioselectivity are likely to be important to commerce, in view of the
increasing trend in marketing single-enantiomer drugs.

UPLC: A MODERN ANALYTICAL TECHNIQUE

 

INTRODUCTION

High-performance liquid chromatography (HPLC) is an important liquid

chromatography (LC) technique that is used for the segregation of different
components in mixtures3. It is also used for the identification and quantification of
compounds in the process of drug development. To achieve the dramatic increase
in resolution, speed and sensitivity in LC, a significant advancement in the
instrumentation and column technology were made. To achieve the above targets,
Waters in 2004, launched and trademarked Ultra Performance Liquid
Chromatography (UPLC) which is based upon small, porous particles (sub
2micron particles). Van Deemter equation is the principle behind this evolution
which correlates the connection between linear velocity and plate height2. The
small particles require a high pressure to work with UPLC i.e., 6000 psi which is
typically the upper limit of conventional HPLCs. It was observed that when the
particle size is decreased below 2.5 µm, there is a remarkable increase in the
effectiveness and this effectiveness increases the linear speed or rate of flow. This
method reduces the mobile phase volume consumption by at least 80% compared
to HPLC with a shorter runtime of about 1.5 min. The smaller sized particles
increase the pressure up to 1000 bars or more which can alone increase the
retention factor of the separation5. Lower injection volume is required for UPLC
which results in higher efficiency and increase in resolution. The higher column
temperature reduces the mobile phase viscosity resulting in the high diffusion
coefficient and flow rate without significant loss in efficiency and increase in
column back pressure. The cost and make the technology is environment friendly6.

CHEMISTRY OF SMALL PARTICLES

Smaller particles provide not only increased efficiency, but also the ability to work
at increased linear velocity without a loss of efficiency, providing both resolution
and speed. Efficiency is the primary separation parameter behind UPLC, since it
relies on the same selectivity and retentivity as HPLC. In the fundamental
resolution (Rs) equation: resolution is proportional to the square root of N3.

But N is inversely proportional to particle size (dp): as the particle size is lowered
by a factor of three. For example, 5 µm (HPLC scale) to 1.7 µm (UPLCscale), N is
increased by three and resolution by the square root of three or 1.7. N is also
inversely proportional to the square of the peak width.

                    

This illustrates that the narrower the peaks are, the easier they are to separate from
each other. Also, peak height is inversely proportional to the peak width.

                    

So as the particle size decreases to increase N and subsequently Rs, an increase in

sensitivity is obtained, since narrower peaks are taller peaks. Narrower peaks also
mean more peak capacity per unit time in gradient separations, desirable for many
applications, e.g., peptide maps. Still another equation comes into play when
migrating toward smaller particles3.

               

This relationship also is revealed from the van Deemter plot. As particle size
decreases, the optimum flow F to reach maximum N increases. But since back
pressure is proportional to flow rate, smaller particle sizes require much higher
operating pressures. A system that can both reliably deliver the requisite pressures
and that can maintain the separation efficiency of the small particles with tightly
managed volumes. Efficiency is proportional to column length and inversely
proportional to the particle size.

                             

Therefore, the column can be shortened by the same factor as the particle size
without loss using a flow rate three times higher due to the smaller particles, the
separation is completed in 1/9 the time while maintaining particles areresolution.
Although high efficiency, nonporous 1.5- commercially available, they suffer from
poor loading capacity and retention due to low surface area. Silica-based particles
have good mechanical strength but can suffer from a number of disadvantages,
which include a limited pH range and tailing of basic analytes. Polymeric columns
can overcome pH limitations, but they have their own issues including low
efficiencies, limited loading capacities and poor mechanical strength. Packed bed
uniformity is also critical, especially if shorter columns are to maintain resolution
while accomplishing the goal of faster separations7.

PRINCIPLE

The principle of UPLC is based on the Van Deemter relationship which explains
the correlation between flow rate and plate height. The Van Deemter equation
shows that the flow range with the smaller particles is much greater in comparison
with larger particles for good results

                           

Where H represents height equivalent to the theoretical plate (HETP), A, B & C

are the constants and v is the flow rate (linear velocity) of the mobile phase. The
aim is to minimize HETP to improve column efficiency8. The term A does not
depend on velocity and indicates eddy mixing. It is smaller if the columns are filled
with small and uniform sized particles. The term B denotes the tendency of natural
diffusion of the particles. At high flow rates, this effect is smaller, so this term is
divided by v. The term C represents the kinetic resistance to equilibrium during the
process of separation. The kinetic resistance is the time lag that involved in moving
from the mobile phase to the stationary phase and back again. The higher the flow
rate of the mobile phase, the more molecules on the packing material inclines to
lag behind molecules in the mobile phase. Thus, this term is inversely proportional
to linear velocity. Consequently, it is likely to enhance the throughput, and without
affecting the chromatographic performance, the separation can be speeded up9.
The use of UPLC has necessitated the improvement of existing instrumentation
facility for LC, which takes the benefit of the separation performance (by
decreasing dead volumes) and consistent pressures (about 500 to 1000 bars,
compared with 170 to 350 bars in HPLC). Efficiency is proportionate to the length
of the column and inversely proportional to the radius of the particles.
Consequently, the column length can be reduced by the similar factor as the
particle radius without affecting the resolution3

ADVANTAGES OF UPLC 

1. It is more selective and sensitive with high resolution performance and faster
resolving power. 2. It also reduces process cycle time and assures endproduct
quality with reduced cost of operation and decreased run time1.
3. It increases sensitivity and provides quick analysis through the use of a novel
column material of very small particle size3. 

4. It decreases the consumption of solvent and increases sample throughput and

also provides realtime analysis in step with manufacturing processes4.

DISADVANTAGES OF UPLC

1. Due to increased pressure requires more maintenance and reduces the life of the
columns of this type8.

2. In addition, the phases of less than 2 µm are generally non-regenerable and thus
have limited use10. 

3. Higher price of instruments, spare parts and columns6.

4. Also detector and data collection system (CDS) may not cope with sharper
peaks (data acquisition rate). 

5. So far only binary pump systems (not ternary or quaternary). This may make
method transfer not straightforward9.

Sample Injection

The use of the injector is to add precisely measured, a small volume of solution
containing the sample in the mobile phase. The injection must be done
reproducibly and accurately. Conventional injection valves may be manual or
programmed and to guard the column from extreme pressure instabilities, the
injection process must be comparatively pulse-free. To reduce the potential band
spreading, the swept volume of the device is desired to be minimal. A quick
injection cycle time is required to fully avail the speed afforded by UPLC. To
increase the sensitivity, low volume injections with minimal

carryover are required. The volume of the sample in UPLC is usually 2-5 μl.
Nowadays, direct injection approaches are utilized for the biological samples.

UPLC Column 
Resolution is increased in a 1.7μm particle packed column because efficiency is
better. Separation of the components of a sample requires a bonded phase that
provides both retention and selectivity. Four bonded phases are available for UPLC
separations: 

i. ACQUITY UPLC TM BEH C8 (straight chain alkyl columns)

(ii) ACQUITY UPLCTM BEH C18 (straight chain alkyl columns)

     (iii) ACQUITY UPLC BEH Shield RP18 (embedded polar group column) and 

     (iv) ACQUITY UPLC BEH Phenyl (phenyl group tethered to the silyl
functionality with a   C6 alkyl)

Each column chemistry provides a different combination of hydrophobicity, silanol

activity, hydrolytic stability and chemical interaction with analytes. 

ACQUITY UPLC BEH C18 and C8 columns are considered the universal columns
of choice for most UPLC separations by providing the widest pH range. They
incorporate tri functional ligand bonding chemistries which produce superior low
pH stability. This low pH stability is combined with the high pH stability of the
1.7μm BEH particle to deliver the widest usable pH operating range.

ACQUITY UPLC BEH Shield RP18 columns are designed to provide selectivity
that complements the ACQUITYUPLC BEH C18 and C8 phases.

ACQUITY UPLC BEH Phenyl columns utilize tri functional C6 alkyl tethered
between the phenyl ring and the silyl functionality. This ligand, combined with the
same proprietary end capping processes as the ACQUITY UPLC BEH C18 and C8
columns, provides long column lifetimes and excellent peak shape. This unique
combination of ligand and end capping on the 1.7μm BEH particle creates a new
dimension in selectivity allowing a quick match to the existing HPLC column. An
internal dimension (ID) of 2.1 mm column is used. For maximum resolution,
choose a 100 mm length and for faster analysis, and higher sample throughput,
choose 50 mm column. Half-height peak widths of less than one

second are obtained with 1.7μm particles, which gives significant challenges for
the detector. In order to integrate an analyte peak accurately and reproducibly, the
detector sampling rate must be of high enough to capture enough data points across
the peak. The detector cell must have minimal dispersion (volume) to preserve
separation efficiency. Conceptually, the sensitivity increase for UPLC detection
should be 2-3 times higher than HPLC separations, depending on the detection
technique. MS detection is significantly enhanced by UPLC; increased peak
concentrations with reduced chromatographic dispersion at lower flow rates
promote increased source ionization efficiencies. 

The binary solvent manager uses two individual serial flow pumps to deliver a
parallel binary gradient. There are built-in solvent select valves to choose from up
to four solvents. There is a 15,000psi pressure limit (about 1000 bar) to take full
advantage of the sub-2μm particles. The sample manager also incorporates several
technology advancements. Using pressure assisted sample introduction, low
dispersion is maintained through the injection process, and a series of pressures
transducers facilitate self-monitoring and diagnostics. It uses needle-in-needle
sampling for improved ruggedness and needle calibration sensor increases
accuracy. Injection cycle time is 25 seconds without a wash and 60 sec with a dual
wash used to further decrease carry over. A variety of micro titer plate formats
(deep well, mid height, or vials) can also be accommodated in a thermostatically
controlled environment. Using the optional sample organizer, the sample manager
can inject from up to 22 micro titer plates. The sample manager also controls the
column heater. Column temperatures up to 65°C can be attained. To minimize
sample dispersion, a “pivot out” design allows the column outlet to be placed in
closer proximity.

Different types of columns being used in UPLC are packed with particles which
are produced through different technologies. These are as follows:

1. Charged Surface Hybrid [CSH] particle technology,

2. Ethylene Bridged Hybrid [BEH] particle technology,

3. High Strength Silica [HSS] particle technology and

4. Peptide Separation Technology (PST).

CSH Particle Technology 

CSH Technology is the newest methodology in the development of hybrid

materials which utilizes lowlevel surface charged particles for the enhancement of
the selectivity and sharpness of the peaks. Hybrid based packing material approach
provides sharp peaks especially for basic compounds under low pH with higher
efficiency and chemical stability. CSH C18, CSH Phenyl hexyl, and CSH Fluoro
phenyl are the different types of CSH particles being widely used.

These columns have the advantage of exceptional peak shape, increased loading
capacity (CSH C18); complementary selectivity to straight chain alkyl phases
(CSH-phenyl-hexyl); selectivity for positional isomers, halogenated and polar
compounds (CSHfluoro phenyl). The other advantages include- higher stability at a
wide range of pH, improved batch to batch reproducibility and fast column
equilibration after any change in the pH of the mobile phase.

Applications of CSH technology based columns include the analysis of basic

compounds even in their ionized form. While analyzing the basic compounds
under low pH and reversed phase conditions, poor peak shape and retention often
result. Whereas, CSH Phenyl hexyl columns, provide exceptional peak shape for
basic drugs under acidic mobile phase conditions. 

BEH Particle Technology 

For more than a decade, hybrid particle technology [HPT] has delivered
incomparable versatility and performance, enabling chromatographers to push the
limits of LC separations. The XTerra particle was the first commercially available
option to improve the issues (poor peak shape for basic compounds and column
longevity due to chemical instability) without the drawbacks of unpredictable
selectivity produced by alternative materials such as zirconia, organic polymers,
and graphitic carbon. With the commercialization of 2.5 μm XTerra particles, the
concept of fast HPLC with small particles was born, improving the productivity of
chromatographic laboratories globally.

Straight chain alkyl columns (BEH C18 and C8), embedded polar group column
(BEH Shield RP18) and UPLC BEH Phenyl (phenyl group tethered to the silyl
functionality with a C6 alkyl) are the different types of BEH particle technology
based columns which are being widely used.

These columns provide higher retention, selectivity (C18); exceptional efficiency,

peak symmetry, chemical stability (C8); enhanced peak shape and higher
compatibility with 100% aqueous mobile phase (RP18); chemical stability,
reproducibility and peak shape (phenyl). Applications include the rapid assay of
cytochrome p450 isoenzymes which are responsible for more than 90% of drug
metabolism on the market today. Cytochrome p450 isoenzymes are used to
determine the level of drug inhibition, induction or drug-drug interaction that takes
place. The ultra-low dispersion and system dwell volume of UPLC technology
enables the rapid analysis of these enzymes in less than 30 seconds.

HSS Particle Technology 

HSS particle technology is the advanced technology, born from an innovative

synthetic process in which the mechanical stability is improved while the pore
volumes remain similar to that of HPLC silica-based materials. This results in an
advanced technique which provides higher retention in comparison to the hybrid
particles. HSS T3, HSS C18, HSS C18 SB, HSS PFP and HSS CN are the different
types of HSS particles being widely used.

These columns provide balanced retention of polar and hydrophobic molecule

(T3); exceptional peak shapes, increased retention (C18); greater retention of basic
compounds (CS 18 SB); ideally suited for planar aromatic, positional, halogenated
compounds (PFP); ultra stable retention and compatible with both reversed-phase
and normal-phase techniques (CN).

Applications of this technology include the analysis of tetracycline antibiotics

which are commonly prescribed for the treatment of bacterial infections. The
ACQUITY UPLC HSS C8 column enables a single UV-based method for the
simultaneous separation of oxytetracycline including its degraded and related
products (which are produced due to drug fermentation) as well as additional
veterinary antibiotics

PST Columns 

PST utilizes the C18 BEH Technology  particles whose particles sizes range from
1.7 µm to 10 µm and the column dimension ranges from 75 µm to 30 mm internal
diameter (i.d) and column length from 50 mm to 250 mm. They are used in all kind
of research and development that involves analysis and isolation of peptides. The
PST columns provide sharp symmetrical peaks.

Solvent Delivery System

The solvent delivery system must perform reproducible high pressure pumping
with a smooth and constant flow of solvents. UPLC systems routinely operate at
8000-15000 psi. The delivery system must also remunerate for a variety of solvents
used in an isocratic, linear & nonlinear gradient elution and solvent compressibility
for a wide range of pressures. The Acquity UPLC binary solvent manager has two
solvent delivery modules operating in parallel for high pressure merging of two
solvents in <140 μL internal system volume. The dissolved gases are removed by
vacuum up to four eluents plus two wash solvents. 

The Detector

The detector employed for the UPLC should be able to give a high sampling rate
with narrow obtainable peaks (<1 s half-height peak width) and the dispersion of
the peaks should be minimum so that the wastage of the separated solute is less on
the column. The UPLC technique provides the sensitivity of separation two to
three times more than the 

previous analytical method HPLC, which is also due to the method employed for
the detection. The  detectors employed in the UPLC are Acquity photodiode array
(PDA) and Tunable Vis-UV (TUV) in which Teflon AF is used which provides an
internally reflective surface and enhances the light transmission efficiency by
eliminating the internal absorptions. These have path lengths 10 nun, acquisition
rates 20 (PDA) and 40 (TUV) points, and total internal volume 500 nL. Mass
spectrometric detection has also been used with UPLC.

Applications of UPLC

Analysis of natural products and traditional herbal medicine

These technique is popularly use for the separation of natural products and
traditional herbal medicine. It has a highly advanced detection and separation
capabilities to identify active compounds that are presents in the samples of natural
products and herbal medicines.

Study of metabonomics /metabolomic

Metabonomics studies are carried out in labs to accelerate the development of new
medicines. It provides a quick and robust method for detecting the changes,
improves understanding of potential toxicity, and allows observing the capacity.
The correct application of metabolomics and metabolomic information helps in the
discovery, development, and manufacturing processes in the biotechnology and
chemical industry companies.
Identification of metabolite

Biotransformation of new chemical entities (NCE) is necessary for drug discovery.

When a compound reaches the development stage, its identification becomes a
regulated process. UPLC addresses the complex analytical requirements of new
discovery by

providing unmatched resolution, sensitivity, and mass accuracy.

ADME (Absorption, Distribution, Metabolism, Excretion) Screening:

Pharmacokinetics studies include studies of ADME. It studies important physical

and biochemical properties like absorption, distribution, metabolism, elimination,
etc. where such compounds show its activity against the target disease.

Manufacturing / QA / QC

Identification of purity, quality, safety and efficacy are the most important factors
that need to be considered while manufacturing a drug product. For the successful
production of quality pharmaceutical products, the raw materials need to meet the
purity specification. These can be achieved with the help of UPLC technique.

Impurity profiling

These techniques easily detect the impurities present if it is presents in very trace
levels too. UPLC combines with same mass.LC/MS, which by running with
different low and high collision energies, has been successfully used for the
detection of drug and endogenous metabolites.

Improved Resolving Power in Peptide Maps 

Peptide mapping is an essential technique for the characterization of proteins. Due

to exceptionally reduced instrument and column dispersion, the analyzes of tryptic
digest of phosphorylase by UPLC technology provides significantly improved
resolution, peak capacity, and sensitivity compared to HPLC, allowing the detailed
characterization of the protein. Multi-Residue Analysis of Pharmaceuticals in
Waste Water 
The water used in the pharmaceutical companies is found to have the traces of
various cholesterollowering statin agents, anti-ulcer agents, antibiotics, beta-
blockers, analgesics, antiinflammatory agents, lipid regulating agents, psychiatric
drugs, and histamine H2 receptor antagonists. UPLC coupled with Q-TOF-MS is
used to confirm and screen these drugs in the samples of waste water treatment
plant.

Method Development / Validation  

Method development and validation is a complex process and consumes a lot of

time. For the development of a robust and reliable method, many combinations of
different parameters e.g. mobile phase, temperature, pH, column and gradient
chemistry etc. UPLC is an important method used in the laboratory which reduces
the cost and increases the efficiency of analysis required for developing and
validating the method. With UPLC, the speed of the separation increases and
efficiency improves, which results in the fast development of methodologies. High
stability of the UPLC columns provides the possibility of selection of column
temperature and pH from a wide range.  

Forced Degradation Studies (FDS)  

Forced degradation/ stress testing is performed under accelerated environment. The

experimental conditions cause the candidate compound to degrade under extreme
conditions like acid and base hydrolysis, peroxide oxidation, photo-oxidation and
thermal stability to identify the resultant degradation products. Use of UPLC with
photodiode array and MS analysis supports the identification of degradation
products and also reduces the time needed to evolve stability indicating methods.

CONCLUSION:

UPLC decrease the analysis time, which in turn reduces consumption of solvent
that plays a vital role in analytical method development. It also facilitates the
analysis of complex mixtures in relatively short time and the peaks obtained with
the help of this method are clearer than that of HPLC. UPLC increases productivity
in both chemistry and instrumentation by providing more information per unit of
work as it gives increased resolution, speed, and sensitivity for liquid
chromatography. The UPLC column even can withstand high back up pressure.