High Pressure Liquid Chromatography  '!

where separation occurs. Since the stationary phase is composed of micrometer size porous
particles, a high pressure pump is required to move the mobile phase through the column. The
chromatographic process begins by injecting the solute onto the top of the column. Separation
of components occurs as the analytes and mobile phase are pumped through the column.
Eventually, each component elutes from the column as a narrow band (or peak) on the
recorder.

Fig. 13. Functional schematic of a modern HPLC instrument.

Detection of the eluting components is important, and this can be either selective or
universal, depending upon the detector used. The response of the detector to each component
is displayed on a chart recorder or computer screen and is known as a chromatogram. To
collect, store and analyze the data, computers, integrators and other data processing
equipment are frequently used.

CONSTANT PRESSURE PUMPS

The primary advantages of constant pressure pumps are simplicity and freedom from
pulsations, resulting in smooth baselines. The most simple of these are usually inexpensive,
easy to operate, and easy to maintain. However, they suffer from several disadvantages. Flow
rate must be monitored carefully and constantly, especially when performing either qualitative
or quantitative analysis. Flow rates can change if the solvent viscosity changes due either to a
temperature or composition change. Changes in flow rate can influence both qualitative and
quantitative analysis. In qualitative analysis, component identification is primarily based on
matching retention volumes. If these change as a result of flow rate changes, especially during
the analysis of complex mixtures, component location and identification becomes difficult. In
quantitative analysis, the most common detectors (UV and RI), are concentration sensitive.
Changes in flow rate affect the time that a chromatographic band passes through the detector
cell, which in turn show up as changes in peak area, which is used for quantitation.
 '" Cellular and Biochemical Sciences

CONSTANT FLOW PUMPS

Constant-flow systems are generally of two basic types: reciprocating piston and positive
displacement (syringe) pumps. The basic advantages of both systems are their ability to repeat
elution volume and peak area, regardless of viscosity changes or column blockage, up to the
pressure limit of the pump.
Reciprocating piston pump can maintain a liquid flow for indefinitely long time, while a
syringe pump has to be refilled after it displaces the whole syringe volume. On the other hand,
a syringe pump does not have any flow and pressure pulsations compared to the reciprocating
pump. For the micro-HPLC applications a syringe pump allows for the maintaining of a
constant flow at the microliter per minute flow rate range.

RECIPROCATING PISTON PUMPS

The basic principle of the reciprocating single piston pump is shown in Fig. 14. The piston
expels liquid through a one-way valve (check valve). The pumping rate is usually adjusted by
controlling the distance the piston retracts, thus limiting the amount of liquid pushed out by
each stroke, or by the cam rotating speed. The main disadvantage of this type of pump is
sinusoidal pressure pulsations which lead to the necessity of using pulse dampers.

Fig. 14. Schematic of the reciprocating single piston pump.

DUAL PISTON PUMPS

A more efficient way to provide a constant and almost pulse free flow is the use of dual-headed
reciprocating pumps (Fig. 15). Both pump chambers are driven by the same motor through a
common eccentric cam; this common drive allows one piston to pump while the other is
refilling.
As a result, the two flow-profiles overlap each other significantly reducing the pulsation
downstream of the pump; this is visualized below. Since the acceleration/deceleration profile
is somewhat non-linear, the more efficient types of these pumps use eccentricity-shaped cams
to obtain the best overlapping of the pressure curves and to obtain smooth flow.
The advantages of this pump are the unlimited solvent reservoir allowing long-term
unattended use and quick changeover and clean out capability. However, unless special care
has been exercised in manufacture, these pumps may have several disadvantages. There is a
 High Pressure Liquid Chromatography  '#

Fig. 15. A dual-head reciprocating pumps.

tendency for the incompletely compensated pulsations to be observable at high refractive

index detector sensitivities, especially at low flow rates where piston cycles are widely spread.
Furthermore, since each head has two check valves, pump reliability depends on the
cleanliness of the mobile phase and continued sealing capability of four check valves on each
cycle, with cycles normally occurring several times per minute. Recent improvements to this
popular pumping system include:
A computer-designed camshaft is used to achieve maximum overlap of pump strokes,
resulting in virtually undetectable pulsation or ripple.
Staggered inlet/outlet lines are employed to allow complete flushing when liquids are
changed or if air is inadvertently drawn through the pump.
Small-volume check valves are used to allow the pumps to function reliably at flow
rates as low as 0.001 mL/min. This has the added benefit of providing excellent
gradient reproducibility especially when programs start from extremely low
concentrations.
There are fewer moving parts, with all maintenance-requiring components (pump
seals, check valves) readily accessible from the front of the instrument.
A wide flow rate range (0.01 to 10 ml/min) is provided without gear change.
Check valves on the reciprocating pump are the weakest part. It may be easily
contaminated or clogged which leads to the pump malfunction. Most of the recent HPLC
instruments use improved dual piston pumps which have three or even two check valves.
The first piston, called low pressure, is sucks the liquid from the reservoir while the second
(high pressure piston) is supplying the eluent to the system. Then the first piston refills the
second piston very fast, during 1/100 of the whole pump cycle. This scheme allows the use of
only 3 check valve, one of which is working under low pressure. There are no cavitations
effects. Because the piston volumes are small (~100 l), pressure pulsations are small and sharp
and easy to damp. The schematic of this pump is shown in the Fig. 16.
Another type of dual piston pump uses only two check valves, but piston volumes are
different. While the smaller piston dispenses an eluent in the HPLC system, the bigger piston is
 '$ Cellular and Biochemical Sciences

Fig. 16. Dual piston pump.

sucking an eluent. When pistons change their direction, the bigger piston simultaneously
refills the smaller chamber and dispenses eluent into the system. This set-up allows only two
check valves for the dual piston pump.

GRADIENT ELUTION
Blending and Mixing Mobile Phase Components
In many of the modes of modern liquid chromatography, it becomes readily apparent that the
most practical system for the delivery of the mobile phase is that which can combine several
liquids in different proportions at the command of the operator. This blending capability
greatly speeds the process of selecting the optimum eluent mixture required for isocratic
analysis and becomes absolutely necessary when programming the mobile phase composition
(gradient elution). These techniques have been discussed earlier from the point of view of
chromatography; now we shall treat them from the point of view of instrumentation. The two
primary methods of blending the mobile phase components are known as high pressure
mixing and low pressure mixing.

HIGH-PRESSURE MIXING
In high-pressure mixing systems, individual high pressure pumps are used to provide each
liquid. The outlet of each pump is either connected to a mixing connector (usually referred to as
a T since there are normally two inlet lines and one outlet line) or to a mixing chamber. In
these, the two flows are blended en route to the injector and column. In other words, mixing is
accomplished on the high-pressure side of the pumps. Most modern liquid chromatography
systems utilizing this principle include pump-drive electronics which control the pumping
speed. Blending is achieved through adjustment of a master controller. The flow rate which
always represents the combined (total) output of both pumps is selected and then the dividing
control adjusted to provide the desired ratio of the two components. These controls are
commonly calibrated to read out the percentage of strong component in the mixture. The
stronger component is considered Component A; thus, the mixing control is labeled %A in
A+B. When a given %A is dialed in, the mixing electronics automatically divides the output
signal between the two pumps.
 High Pressure Liquid Chromatography  '%

Fig. 17. Mixing of two mobile-phase components.

Pump A (strong component) receives the percentage of the signal called for in the control
indicator, and Pump B (weaker component) receives the remainder of the signal, so that the
total output of both pumps always equals the pre-selected flow rate. Fig. 17 shows the mixing
of two mobile-phase components. It is also possible to prepare ternary mobile phases by
utilizing three separate pumps. Each of these pumps will then deliver a separate solvent into
the mixing chamber.
Among the advantages of high-pressure mixing is the ability to achieve precise control and
repeatability of mobile phase mixtures to within 0.1% levels (only within the medium eluent
composition range), rapid response to changes in concentration and the ability to utilize each
pump separately if separate isocratic pumping systems are required. However, there are some
disadvantages for this type of gradient formation:
Even special mixing chambers do not provide complete mixing;
Many HPLC solvents being mixed will change the total volume, sometimes up to 20%;
Mixing accuracy within the 0-10% range for component A or B is usually poor;
High pressure mixing needs two or sometimes three high pressure pumps, which
increase the cost of the whole system.

Low-Pressure Mixing
In low-pressure systems, mixing is accomplished prior to the pump, at its low-pressure side
and the overall flow rate is controlled by a single pump. Blending of the mobile-phase
components is accomplished with controls which are calibrated in somewhat the same manner
as those used for high-pressure mixers that is, controlling the percentage of the strong
component though some systems allow the mixing of three liquids. In general, two types of
blending systems are used.
In the first case, proportioning valves, normally solenoid operated, are used to deliver the
individual liquids. The controller simply divides the signal according to the percentage of each
component and each valve is opened for the proper period of time. Usually the valves deliver
the individual liquids into a mixing chamber which then feeds the blend to the pump. In some
systems, the mixing chamber may be missing and the valves feed the mobile-phase
components through a mixing connector directly to the high-pressure pump.
 '& Cellular and Biochemical Sciences

The primary advantage of such systems is that they offer the ability to blend two or more
liquids without the increased expense and maintenance of additional pump(s).This is now the
most popular scheme of gradient formation, although the precision of eluent composition for
medium concentration range is better for the high pressure mixing.

PROGRAMMING THE MOBILE PHASE COMPOSITION

In gradient elution, the concentration of the mobile phase is varied during a run. There are two
possibilities: the change in concentration is linear with time or it is not linear. In the latter case,
the plot describing the program might be convex or concave. Instruments which are capable of
mixing liquids for the mobile phase are also capable of carrying out gradient elution
automatically.
Each step of the linear program is described by four variables the initial and final
concentration (%) of Component A in the mixed mobile phase, the rate of program (%/min)
and the length (time) of the program. Three of these four fully describe the program; in practice,
the instruments permit the setting of the two concentrations and either the time or the rate of
the program for each step. The electronic control system will then automatically set either the
rate or the time of the program. The situation with curved gradient profiles depends on the
particular instrument. Some less convenient programmers require step-by-step entry of
multiple percentage vs. time plateaus to approach curved gradient profiles while in more
sophisticated systems, one can select from a number of programmed profiles.
In the actual instruments, one has to set the initial and final concentrations, the time of the
program and select which program profile is desire. This is done by certain keys corresponding
to the various exponent values. Naturally, in sophisticated instruments where various
programs can be combined, curved programs can also be included together with linear
programs and isocratic periods. The most important consideration in gradient elution is that
the whole program a simple as well as a complex multistage program can be exactly
reproduced upon command since inaccurate gradient repetition can contribute to significant
qualitative and quantitative errors.

PROBLEMS IN PUMPING LIQUIDS

The common problems encountered in pumping mobile phases are solvent degassing,
corrosion, and compressibility. Compressibility has already been discussed and since its
influence is minor, we shall not deal with it further. The two other problems, however, need
further analysis.

DEGASSING
As previously noted, when the mobile phase contains excessive gas which remains dissolved at
the pressure produced by the column, the gas may come out of the solution at the column exit
or in the detector, resulting in sharp spikes. Spikes are created by microscopic bubbles which
change the nature of the flowing stream making it heterogeneous while drift may occur as
these microscopic bubbles gradually collect and combine in the detector cell. The main problem
is oxygen (from the air) dissolving in polar solvents, particularly water. If needed, degassing
may be accomplished by one of the following methods or their combination:
 High Pressure Liquid Chromatography  ''

Subjecting the liquid to vacuum

Heating the liquid until boiling occurs.
Placing the container of liquid in an ultrasonic bath or inserting an ultrasonic probe in
it.
Bubbling a fine stream of helium through the liquid; helium has the unique ability to
spurge other gases out of solutions.
The best results were found by applying vacuum to each solvent for about 5 min, with
subsequent helium purging and storing under a helium atmosphere.

CORROSION
Most commercially available instruments are constructed of Type 316 stainless steel. This
material represents the best compromise of corrosion resistance, workability, strength, and
cost. It is inert to all bases, all organic liquids and most non-halogenated acids at pH values
above 2.0. Type 316 stainless steel is, however, extremely vulnerable to contact with
halogenated acids, like HCl, even at concentrations as low as 0.01 normal. When attempting to
convert a thin-layer or (glass) column chromatographic method to HPLC, substitute another
acid such as nitric, boric, or acetic for halogens. Ion-exchange systems should be monitored
carefully for discolored eluent especially when small particle packings that exhibit high back
pressure (2500+ psi) are used, since several researchers have observed highly accelerated
corrosive reactions at these pressures with some buffer systems.

INJECTORS
Injectors for liquid chromatographic systems should provide the possibility of injecting the
liquid sample within the range of 0.1-100ml of volume with high reproducibility and under
high pressure (up to the 4000 psi). They should also produce minimum band broadening and
minimize possible flow disturbances. Generally, the most useful and widely used sampling
device for modern LC is the micro-sampling injector valve (Fig. 18).

Fig. 18. Rheodyne injector.

Because of their superior characteristics valves are now used almost to the exclusion of
syringe injection. With these sampling valves, samples can be introduced reproducibly into
pressurized columns without significant interruption of flow, even at elevated temperatures.
Fig. 19 shows schematic drawings of a six-port Rheodyne valve in which the sample fills an
external loop.
Compared to shorter, wider i.d. sample loops, long, narrow loops are preferred when large
sample volumes are required, because of lesser band-broadening effects. Alternatively, a
specially designed syringe may be used to inject a small volume (e.g., <10, ul) into the loop
! Cellular and Biochemical Sciences

Fig. 19. Six-port Rheodyne valve.

when required, although in this case the precision in the sample introduction is dependent on
the precision of syringe delivery.
A clockwise rotation of the valve rotor places the sample-filled loop into the mobile-phase
stream with subsequent injection of the sample onto the top of the column through a low-
volume, cleanly swept channel. Other valve types (e.g., Siemans and Valco) use an internal
sample cavity consisting of an annular groove on a sliding rod that is thrust into the flowing
stream. The minimum injection volume which can be made with the valve-type injectors is 60
nl.
Valve injection allows the rapid, reproducible, and essentially operator-independent
delivery of a wide range of sample volumes (e.g., from 60 nl up to several milliliters) at
pressures up to 7000 psi with less than 0.2% error. High-performance valves provide extra
column band-broadening characteristics comparable or superior to that of syringe injection.
Manually operated valves are only moderately expensive, and automated versions can be
obtained at somewhat higher cost. A minor disadvantage of most sample valves is that the
sample loop must be changed to obtain various sample volumes, but this can often be achieved
in a few minutes. Another advantage of sampling valves is that they can be located within a
temperature-controlled oven for use with samples that require handling at elevated
temperatures (0 150C).
Low-volume switching valves are also available (e.g., Valco, Rheodyne, Siemans) for use in
special techniques such as recycle chromatography and column switching. Some of these
valves can be operated at pressures up to 7000 psi, and often they can be used at elevated
temperatures. The more common valves can be obtained in 3-, 4-, 6-, 8-, or 10-port
configurations, for use in either the manual or automated mode.

AUTOMATIC INJECTORS
With commercially available automatic sampling devices, large numbers of samples can be
routinely analyzed by LC without operator intervention. Such equipment is popular for the
analysis of routine samples (e.g., quality control of drugs), particularly when coupled with
automatic data-handling systems. Automatic injectors are indispensable in unattended
 High Pressure Liquid Chromatography !

searching (e.g., overnight) for chromatographic parameters such as solvent selectivity, flow-
rate, and temperature optimization.
Most of the auto-samplers have a piston metering syringe type pump to suck the pre-
established sample volume into a line and than transfer it to the relatively large loop (~100 ml)
in a standard six-port valve. The simplest auto-samplers utilize the special vials with pre-
suarization caps. A special plunger with a needle, push the cap down to the vial and displace
the sample through the needle into the valve loop. Most of the auto-samplers are
microprocessor controlled and can serve as a master controller for the whole instrument
(Fig. 20).

Inject
To column

From pump

op
Lo

Waste

Fig. 20. Injection of sample.

COLUMN HARDWARE
Today, column material is normally Type 316 stainless steel, once again chosen because it
offers the best compromise of cost, workability, and corrosion resistance. Most commercial
columns available today have internal diameters of either 2.6-3 mm or 4.6-5 mm (Fig. 21). Most
leading manufacturers obtain a great amount of interchangeability between column sizes
without excessive fitting replacement by supplying columns with a variety of internal
diameters, but a uniform in. outside diameter. Naturally, preparative columns have larger
diameters: usual O.D. values are 3/8 - 5/8 in., corresponding to internal diameters of 6.4-12.7
mm, respectively.
Most modern liquid chromatography plumbing is performed with compression fittings
such as Swagelok, Parker-Hannifin, Gyrolok, etc. These offer a great deal of flexibility, can be
connected and disconnected frequently without loss of sealing power, are commonly available,

Fig. 21. Standard HPLC column.

! Cellular and Biochemical Sciences

and may be used without special seating and flanging tools allowing users to install, maintain,
modify, and improvise equipment. Great care should be taken in the choice of end fittings,
connectors, and unions however, since inclusion of rapid diameter changes corners and other
unswept areas in a system can destroy a good separation. It is best to obtain the above parts
from a reputable liquid chromatograph manufacturer since most of these companies offer
fittings which are specifically made for LC.
Many reducing unions and column end fittings incorporate special devices designed to
hold packings in place and/or prevent their flowing into the detector or detector tubing. The
most frequently used of these consist of fine porous stainless steel fritted filter discs, usually
with an average opening of 1 mm. These may be forced into the ends of columns or may be in
the connector or end fitting. The important point is that the frit disc must be tightly fitted to
avoid occurrence of large-diameter spaces at the edge where the disc contacts the wall. An
additional benefit of the frit is derived from the fact that it fills what otherwise might be a large
empty area in conversion zones thus; the sample cannot diffuse, cross mix, or undergo other
efficiency-killing effects when passing through these areas.

CONNECTOR TUBING
Connector tubing must be of low cross section to hold separated components in narrow bands
as they are transferred from column-to-column, from column-to-detector, or from detector-to-
detector when mounted in series. Tubing having an internal diameter in excess of 0.634 mm
(0.025 in) can allow peak mixing and band broadening and it is good practice to use tubing no
larger than 0.12 mm (0.005 in) in areas where sample is to be transported.

LINE FILTERS
A line filter should be used between the pump and the sample injector to prevent particulates
from clogging the column inlet. Porous stainless steel filters having a porosity of about 2 m are
typically used in commercial instruments; however, 0.5-mm porosity filters (e.g., Alltech
Associates) are desirable with columns of less than 10-mm particles. To facilitate solvent
changeover, the volume of the line filters should be small.

PRESSURE MEASUREMENT
Pressure monitors are used in the LC equipment as diagnostic tools to optimize separation and
to indicate system problems (e.g., plugging or leaks). Diaphragm or Bourdon-type gauges are
simple, inexpensive, and generally robust. On the other hand, strain-gauge pressure
transducers are more precise and have a smaller internal volume, which facilitates the
changing of solvents. In addition, straingauge types are available with high- or low-pressure
alarms or cut-off circuits, to protect the pump against high-pressure overload by column
plugging or the instrument against fire due to solvent leaks (low-pressure alarm).

COLUMN THERMOSTATS
It often is advantageous to run ion-exchange, size-exclusion and reverse-phase columns at
higher temperatures, and to precisely control the temperature of liquid-liquid columns.
 High Pressure Liquid Chromatography !!

Therefore, column thermostats are a desirable feature in modern LC instruments. Generally,

temperature variation within the LC column should be held within +0.2C. Maintaining a
constant temperature is especially important in quantitative analysis, since changes in
temperature can seriously affect peak-size measurement. It is sometimes important to be able
to work at higher temperatures for the size-exclusion chromatography of some synthetic
polymers because of solubility problems, but precise temperature control is not so important in
this case. High-velocity circulating air baths are most convenient in LC (as in GC). These
devices usually consist of high-velocity air blowers plus electronically controlled thermostats,
with configurations similar to those used in gas chromatographs. Alternatively, LC columns
can be jacketed and the temperature controlled by contact heaters or by circulating fluid from a
constant-temperature bath. This latter approach is practical for routine analyses, but is less
convenient when columns must be changed frequently.

FRACTION COLLECTORS
Since separations are accomplished in minutes, fraction collectors are usually not needed in
modern LC. Manual collection is normally used, and many commercial instruments have
convenient sample-collection ports on the outlet of the detector. Fraction collectors are
sometimes used in preparative chromatography and in conventional size-exclusion
chromatography with larger-diameter columns, because separations are usually much slower.

PRESENTATION OF RESULTS
Chromatographic data are presented almost universally in the form of a chromatogram using
chart recorder. For quantitative analysis and greater precision in retention time measurements,
digital integrators, computing integrators, and computing systems may be employed. Their
specification is essentially fast response time, wide linear dynamic range, and capable of
accepting both narrow (fast eluting) and wide (slow-eluting) peaks. For maximum
convenience, chart recorders should be provided with a wide range of chart speeds, as some
chromatographic methods take but a few minutes to complete whereas others take hours.
In quantitative work, computing integrators and dedicated computers are becoming
popular, for once the detector response data and other basic information have been fed into the
system, and the analytical results are calculated and printed in report form by the computer. In
laboratories where numerous repetitive samples are analyzed these systems can offer a
significant reduction in time and effort made by the operator.

APPLICATIONS
It is quite remarkable to what extent the technique has been successfully applied. In fact, with
laboratories such as those associated with the pharmaceutical industry, modern instrumental
HPLC has brought about a complete change in the methods of routine quality control analysis,
in most instances with a distinct reduction in analysis time and an increase in the analytical
precision.
New and experienced chromatographers alike will be aware how often, after expending
considerable efforts to develop a separation method, it is found that a similar procedure has
already been achieved elsewhere. Scientific communications have proliferated in recent years,
!" Cellular and Biochemical Sciences

making, perhaps, more difficult the task of effectively covering all possible sources of
information. In present scenario, HPLC has wide applications. Some of important ones are as
following:

IN INORGANIC CHEMISTRY
Although the currently available commercial equipment is rather expensive, the HPLC and its
various techniques have already shown to have wide applications in organic chemistry and in
fact have been the doamin of organic chemistry for a long time. In the last few years it has
actually been recognized that HPLC can equally be utilized for the separation of inorganic
compounds. The limitation of HPLC in inorganic chemistry is that only those compounds are
amenable to HPLC investigations which are soluble in solvents being compatible with HPLC
columns.
There are two classes, of soluble inorganic compounds. First, there are ions (anions and
cations) which are usually soluble in aqueous solutions. Second, there are molecular
compounds which are more readily soluble in organic solvents. Thus, two different branches of
HPLC have come into use in inorganic chemistry. Ion chromatography (IC) is used for the
separation of ions in aqueous solution, while non dissociated molecular compounds are
separated in reversed phase systems using bonded alkyl silica phases and organic eluting
agents such as methanol or acetonitrile (Reversed phase liquid chromatography).

ANALYSIS OF NATURAL AND SYNTHETIC PHARMACEUTICAL DRUGS

HPLC is applied at several different levels in pharmacy and pharmacology. The important uses
of HPLC in these areas are:
(a) In production development and product control.
(b) In isolation of pharmaceuticals.
(c) In raw material control.
(d) In clinical analysis, this closely linked to the study of pharmaceuticals.
The retention properties of 140 synthetic drugs or pharmaceuticals on a 10 mm C-18
reversed phase with various methanol water mixtures under isocratic conditions have been
described. Some of the pharmaceuticals are acetyls, alicyclic acids, ascorbic acid, barbital,
saccharin, caffeine, chlorothiozole, diazepam, sulphacarbamide, sulphacetamide,
sulphadiazine, sulphaguanidinde, sulphathiazole, sulphanilamie, phenyl butazone,
tolazamide and vanillin.
There is no small group of solvents that is suitable for the HPLC separation of all
antibiotics. A number of antibiotics have actually been analyzed and separated by HPLC.
These include amoxicillin, ampicillin, anisomysin, chloramphenicols, cycloheximide,
erythromycin, levorin, neomycin, penicillin, polymyxin, streptomycin, trichloromycin and
vancomycin.
Some other examples of HPLC analysis of various pharmaceuticals are the separation of
antipyrine and benzocaine in ear drops, separation of benzoic acid and salicylic acid in
ointments, separation of epinephrine in pharmaceutical preparations on a cation exchange
column and analysis of insulin, glucagons and somatosatatin etc.
 High Pressure Liquid Chromatography !#

Other pharmaceuticals of importance separated and identified by HPLC are lignans,

anthraquinone derivatives, saponins and sapogenins, arbutin drugs, biogenic amines,
deterpene esters, curcubitacins, gossypol, g-benzopyrone derivatives, khellin and visnagin,
terpenes, etc. A selective treatment of all the pharmaceuticals is not possible here, because of
large number of pharmaceuticals commercially available today.

PSYCHOTROPIC DRUGS IN BODY FLUIDS

The HPLC technique has also been used for the separation and identification of psychotropic
drugs such as benzodiazepine, antidepressants, neuroleptics, phenothiazine, butyrophenones
etc. in patients body fluids (such as urine, plasma, cerebrospinal fluids, mothers milk and
saliva). Since most of the psychotropic drugs are almost completely metabolized, a urinary
assay allows no conclusion regarding the amount of the effective mother substance in the
whole body. Hence estimations in urine are rarely made in connection with psychotropic
drugs. For correlation with therapeutic outcome, however, plasma concentrations are
estimated.
The various benzodiazepines have weakly acidic but markedly basic properties. In order
to extract them with an organic solvent, the pH value of the plasma is altered to about 8 and so
a concentrated buffer is generally added to the plasma before extraction. All common organic
solvents, such as benzene, diethyl ether and chloroform can be used for extraction of
benzodiazepines.
Antidepressants are always extracted under basic conditions. Silica column or C-18
reversed phase columns are used for their separation. Extraction has been carried out with a
number of solvents such as light petroleum, hexane, diethyl ether, 1% isoamyl alcohol, 2%
amyl alcohol in hexane, etc.
Phenothiazine and butyrophenones are usually called neuroleptic drugs. A number of
neuroleptics have been extracted on silica and C-18 reversed phase column and many of the
phenothiazines can be well identified by UV detection.

ANALYSIS AMINO ACIDS AND PROTEIN

HPLC has widely been used in the analysis and separation of a number of amino acids as well
as proteins. Before HPLC, the separation was carried out on a cation exchanger and the
detection was performed in the elute after separation by means of ninhydrin. Refractometric
detection or UV detection at 205 nm is usually used in case of HPLC.
Proteins differ from each other in their molecular size, shape, charge and their
hydrophobic properties. Chromatographic separation techniques have been developed which
separate according to size, charge and hydrophobicity and hence these methods are used with
success for the separation and purification of proteins. The chromatography is possible on C-18
reversed phase. The best separation is obtained at pH 2.
The nutritional need of proteins and amino acids essential to the human diet is well
established. The food industry has developed food products which have amino acid
compositions adequate for nutritional requirements. This involves blending proteins from
different sources or fortifying foods directly with essential amino acids. The conventional
amino acid analyzer, based on ion-exchange chromatography followed by derivatization with
!$ Cellular and Biochemical Sciences

ninydrin, has been used for the determination of amino acids in a variety of proteins
hydrolysates. In addition, a number of HPLC procedures have been developed for the
determination of amino acids. The separation of amino acid derivatives with HPLC is generally
carried out on reversed phase columns with acetonitrite aqueous buffer solvent systems. The
aqueous buffer is used to minimize sample ionization and thus improving the peak shape. Both
variable wavelength UV detectors and fluorescence detectors have been used for amino acid
analysis, depending upon the amino acid derivative used.

SEPARATION OF COAL AND OIL PRODUCTS

The time consuming LC methods have now been replaced almost completely by modern HPLC
method. The complexity of distillation residues and similar products is so large that it is almost
impossible to expect the resolution of individual substances even by making use of high
efficiency HPLC techniques. As a result, detailed discussion of such separations is not possible
in the present Chapter.

CARBOHYDRATES
Carbohydrates are present in foods at nature or supplemented levels. Since carbohydrates do
not absorb strongly in the UV spectral region, the refractive index detector is usually employed
in their separation and analysis. A number of variable wavelength UV detectors at 192 nm have
been employed but with no significant increase in sensitivity. The polar bonded phases, e.g.,
LiChrosorb NH2 have been found to be very effective for the separation of carbohydrates with
acetonitrilewater mixtures as mobile phase. This system has actually been used in the
determination of sugar content of Soyabeen extracts, dairy products and molasses. Increasing
the water content of the mobile phase increases the rate of elution and this system has been
used for the separation of higher carbohydrates found in corn syrups. Elution has been found
in order of increasing glucose chain length. Simple carbohydrates have also been analyzed by
using ion exchange columns loaded with Ca2+ or other metal ions and using water as the
eluting solvent. The components elute in order of decreasing molecular size.

DETERMINATION OF PRESERVATIVES AND ANTIOXIDANTS

Preservatives and antioxidants are the additives which prevent microbial or chemical
deterioration of food products. Most preservatives can be determined directly using HPLC. For
example, sorbic acid in beverages can be determined using a reversed phase column with an
isocratic solvent system. Gradient elution on a reversed phase column is required for resolving
food preservatives including p-hydroxy benzoic acid esters (PHB-esters). Most widely used
food preservatives are sorbic acid, benzoic acid, PHB-methyl ester, PHB-ethyl ester, PHB-
propyl ester, biphenylol, biphenyl etc.
A variety of chromatographic procedures have also been developed for the separation of
antioxidants in foods, but HPLC has only been used in the quantitative determination of nine
most commonly used antioxidants simultaneously without derivatization. A method for
analysis of antioxidants in oils, lards and shortenings requires a liquid-liquid extraction from
hexane oil into acetonitrite, followed by sample extract concentration before separation on a
reversed phase column.
 High Pressure Liquid Chromatography !%

ANALYSIS OF VITAMINS
The HPLC has widely been used for analyzing both water soluble as well as fat soluble
vitamins in a variety of food products, fortified foods and animal feeds. Advantages of HPLC
include direct analysis without derivatization, multiple component determinations and
resolution of isomers. Separation of fat soluble vitamins has been carried out on both silica and
bonded phase columns using gradient elution. The reversed phase column has the advantage
of faster equilibrium and better long term retention behavior.

SEPARATION OF STEROIDS AND LIPIDS

HPLC has successfully been applied to the separation of all classes of steroidal hormones.
Thermally labile steroids, such as corticosteroids and non volatile ones such as steroid
conjugates can however, be analyzed relatively easily.
The development of improved techniques for the analysis of lipids in food products has
become increasingly important with the current emphasis on dietary requirements and
nutritional labeling. Column chromatography was formerly used for the separation of various
non polar lipids, glycolipids and phospholipids. These individual fractions were then spotted
on TLC plates for analysis. HPLC techniques have been developed in order to give greater
selectivity and improved quantitation. For HPLC this means that interaction of the non-polar
chains with a polar stationary phase (e.g. silica) can be exploited to get the separation.

FORENSIC CHEMISTRY
The forensic chemistry in concerned with the processing of disputed questions of fact in
criminal, civil and insurance law, which can be clarified by the application of chemical methods
and knowledge. HPLC is widely used for the detection of a large number of poisons,
intoxicants and addictive drugs etc. HPLC is also of great value in the field of chemical
criminology, which is concerned mainly with residues and traced of the non-living milieu.

REFERENCES AND SUGGESTED READINGS

Brown, P.R. (1972) High Pressure Liquid Chromatography: Biochemical and Biomedical Applications, Academic
Press, New York.
Dunges, W., Naundorf, G. and Seiler, N. (1974) J. Chromatogr. Sci., 12: 655.
Engelhardt, H. (1986) Practices to High Performance Liquid Chromatography, Springer-Verlag, Berlin.
Hamilton, R.J. and Sewell (1982) Introduction to High Performance Liquid Chromatography (2nd edn.)
Chapman Hall, London.
Huber, J.K.F. (1978) Instrumentation for High Performance Liquid Chromatography, Elsevier, Amsterdam.
Junk, G.A., Richard, J.J., Grieser, M.D., Witiak, D., Arguello, M.D., Vick, R., Svec, H.J., Fritz, J.S. and
Calder, G.V. (1974) J. Chromatogr., 99: 745.
Kirkland, J.J. (1973) J. Chromatogr., 83: 149.
Kirkland, J.J. and Synder, L.R. (1979) Introduction to Modern Liquid Chromatography (2nd edn.), John Wiley,
New York.
Knox, J.H. (1982) High Performance Liquid Chromatography, Edinburgh University Press, Edinburgh.
Liteanu,C. and Gocan, S. (1974) Gradient Liquid Chromatography, Ellis Harwood, Chichester.
!& Cellular and Biochemical Sciences

Parris, N.A. (1980) Instrumental Liquid Chromatography (3rd edn.), Elsevier Scientific Publisher Company,
Amsterdam, The Netherlands.
Rhys Williams, A.T. (1980) Fluorescence Detector in Liquid Chromatography, Perkin Elmer, Beaconsfield.
Schmidt, J.A. and Kirkland, J.J. (1971) Modern Practice in Liquid Chromatography, Wiley-Interscience, New
York.
Scott, R.P.W. (1986) Liquid Chromatography Detectors, Elsevier, Amsterdam.
Scott, R.P.W. (1976) Contemporary Liquid Chromatography, Wiley, New York.
Simpson, C.F. (1982) Techniques of Liquid Chromatography, Wiley, Chichester.
Synder, L.R. (1970) J. Chromatogr. Sci., 8: 692.
Synder, L.R. (1974) J. Chromatogr., 92: 223.
Synder, L.R. and Kirkland, J.J. (1974) Introduction to Modern Liquid Chromatography, Wiley-Interscience,
New York.
Synder, L.R. (1978) Principals of Adsorption Chromatography, Marcel-Dekker, New York.

REVIEW QUESTIONS
Long Questions
1. What is HPLC? How is it superior to other techniques?
2. What types of detectors used in HPLC? Discuss briefly the UV detector and RI
detector.
3. Discuss briefly the sample injection system used in HPLC.
4. What are the detectors and their basic requirements?
5. Describe various types of pumps used in HPLC?
6. Discuss the method used in HPLC.
7. What is the effect of temperature and pH in HPLC? What are the disadvantages of an
increase in temperature?
8. What is HPLC? Discuss briefly some important applications of HPLC.
9. Write short notes on:
(a) Mobile phases (b) Stationary phases
(c) Noise and drift

Multiple Choice Questions

1. In chromatography, the process of development is called
(a) Elution (b) Illusion
(c) Gradient elution (d) All the above
2. The fixed phase and moving phase of chromatography is alternatively known as
(a) Mobile phase and stable phase
(b) Moving phase and stable phase
(c) Stationary phase and mobile phase
(d) Stationary phase and moving phase, respectively
 High Pressure Liquid Chromatography !'

3. An in crease in the number of theoretical plates improves

(a) Efficiency (b) Resolution
(c) Both efficiency and resolution (d) None
4. Chromatographic development may be carried out by the following techniques
(a) Elution development and gradient elution
(b) Frontal analysis and elution development
(c) Gradient elution and Displacement development
(d) Elution development, Frontal analysis, Gradient elution and Displacement
development
5. As Rf increases, the retention of solutes
(a) Increases
(b) Decreases
(c) Rf and retention of solutes are not correlative
(d) Depends on type of phase
6. The basis of adsorption column chromatography is
(a) Distribution of a solute between two liquids in a column
(b) Adsorption or partition on thin sheets
(c) Distribution of a solute between a solid and liquid phase on a column
(d) Size of solute
7. The solubility is greater affected by Hydrogen bonds
(a) Dipole interactions
(b) Charge transfer process between solute and solvent molecules
(c) All the above
8. What is the general adsorption sequence of functional groups according to increasing
polarity?
(a) Acids and base, aldehydes, amino groups, saturated hydrocarbons
(b) Amino groups, aldehydes, acids and base, saturated hydrocarbons
(c) Saturated hydrocarbons, amino groups, aldehydes, acids and base
(d) Aldehydes, saturated hydrocarbons, amino groups, acids and base
9. How to degas chromatographic solvents?
(a) Subject the solvent to vacuum for 5-10 mins. to remove the gases
(b) Subject the solvent to ultrasonics for 10-15 mins. to remove the gases
(c) Sparse the solvent with a gas that has a very low solubility compared to the oxygen
and nitrogen from the atmosphere
(d) All the above.

Answers
1. (a) 2. (c) 3. (c) 4. (d) 5. (b) 6. (c)
7. (d) 8. (a) 9. (d) 10. (a)