There are several sophisticated analytical methods that are used most abundantly for the precise
quantitative methods microbial assays, such as :
(a) High Performance Liquid Chromatography (HPLC),
(b) Reverse-Phase Chromatography (RPC), and
(c) Ion–Pair (or Paired-Ion) Chromatography,
These three chromatographic techniques shall now be discussed briefly in the sections that follows
:
High Performance Liquid Chromatography [HPLC]
Preamble : Giddings* (1964) rightly predicted that the careful and meticulous application of
relatively ‘small particulate matter’ under the influence of excessively enhanced flow pressure could
definitely improve upon the performance of ‘Liquid Chromatography’ significantly ; and ultimately
one could easily, accomplish an appreciably high number of ‘theoretical plate numbers’. Towards the
later half of 1960s world’s two eminent scientists, Horvath and Lipsky at Yale University (USA), came
forward with the first ever HPLC, and named it as ‘high pressure liquid chromatography’. Nevertheless,
the early 1970s the world witnessed the ever glorious technological supremacy by producing and
using very small silanized silica particles that gainfully permitted the usage of small-volume longer
columns absolutely urgent and necessary to yield the much desired high-resolution performance. In
fact, the latest HPLC is, therefore, commonly known as the ‘high-performance liquid chromatography’
across the globe.
Principles : The particle size of the stationary phase material predominantly plays an extremely
vital and crucial role in HPLC. In actual practice, high-efficiency-stationary phase materials
have been duly researched and developed exclusively for HPLC with progressively smaller partricle
size invariably known as ‘microparticulate column packings’. These silica particles are mostly uniform,
porous, with spherical or irregular shape, and with diameter ranging betwene 3.5 to 10 μm.*
The bonded-phase supports normally overcome a good number of cumbersome and nagging
serious problems that are invariably encountered with the adsorbed-liquid phases. Thus, the molecules
containing the stationary phase i.e., the surfaces of the silica particles are covalently bonded upon a
silica-based support particle.
Example : Siloxanes are duly formed by heating the silica particles in diluted acid for 24–48 hrs.
in order to give rise to the formation of the reactive silonal moiety as depicted below :
When such microparticulate-bonded-phases are compactly packed into a column, the tiny size
of these particles affords a substantial resistance to the ensuing solvent flow ; and, therefore, the mobile
phase has got to be pumped via the column at a flow rate ranging between 1 to 5 cm3 . min– 1.
Advantages of HPLC : The advantages of HPLC are as stated below :
(1) Highly efficient, selective, and broad applicability.
(2) Only small quantum of sample required.
(3) Ordinarily non-destructive of sample.
(4) Rapidly amineable and adaptable to ‘Quantitative Analyses’.
(5) Invariably provide accurate, precise, and reproducible results.
HPLC-Equipments : Modern HPLC essentially comprises of seven vital components, namely :
(a) solvent reservoir and degassing system, (b) pressure, flow, and temperature, (c) pumps and sample
injection system, (d) columns, (e) detectors, (f) strip-chart recorder, and (g) data-handling device and PCbased
control.
Fig. 10.5 represents the HPLC chromatogram of peritoneal (PT) fluid from a subject having an
impaired renal function to whom ‘Cefotaxime’, an antibiotic has been administered intraperitoneally.
Cefotaxime (CTX) gets metabolized to microbioligically ‘active’ and ‘inactive’ metabolites.
PT Fluid : Peritoneal Fluid
DACM : Desacetyl Cefotaxime (Active)
CTX : Cefotaxime
UP1 and UP2 : Two microbiologically inactive metabolites
Reverse-Phase Chromatography [RPC]
The Reverse-Phase Chromatography (RPC) or Reversed-Phase HPLC (RP-HPLC) is
invariably employed for the separation of organic compounds.
In RPC, specifically a relatively nonpolar stationary phase is employed along with such polar
mobile phase as :
methanol, acetonitrile, tetrahydrofuran, water, or
mixture of organic solvents and water.
Organic Solvent—the organic solvent is usally termed as the ‘modifier’ e.g., acetonitrile.
Water—Water content is mostly varied according to the required polarity.
Methanol—It is used for acidic compounds.
Acetonitrile—It is employed for basic compounds.
Tetrahydrofuran (THF)—It is usually used for those compounds having large dipoles comparatively.
In fact, most of these solvents do have low viscosity and are UV-transparent.
Bonded Phases—The abundantly used bonded phases are :
n-Octyldecyl (i.e., C-18 chain),
n-Decyl (i.e., C-8 chain), and
Phenyl Moieties
Polar-Reversed Phase Columns— The polar-reversed phase columns essentially are
polyethylene glycol (PEG) which contain either moieties that interact with polar analytes e.g., phenolic
compounds, multiaromatic ring systems, and hydroxyl-containing compounds.
Ion-Pair (or Paired-Ion) Chromatography
Importantly, perhaps the most valuable of the secondary equilibria variants usually encountered
in the ‘pharmaceutical analysis’ being the ion-pair formation, that may be adequately expressed
for a reversed-phase LLC-System
It has been duly observed that the ion-pair AB thus formed is capable of partitioning very much
into the ensuing stationary phase. However, in many instances the ions A+ and B– fail to do so by virtue
of the fact that their ultimate polarity gain entry into the stationary-phase gradually thereby the evolved
chromatographic resolution is controlled exclusively by the so called ion-pairing phenomenon.
It is, however, pertinent to state here that one may invariably come across a host of ‘drug substances’
that are either acidic or basic in character ; and, therefore, they may be duly rendered into ionic
by carefully regulating the pH of the ensuing mobile phase. In short, ion-pair chromatography possesses
an enormous applicability in the separation of drug substances.
Examples : A few-typical examples pertaining to the ion-pair chromatography are as described
under :
(1) Separation of Niacin, Niacinamide, Pyridoxine, Thiamine and Riboflamin. The admixture
of five vitamins can be separated effectively by making use of the
sodium hexanesulphonate as the ion-pairing agent, on a C—18 column
i.e., ODS-column
(2) Antihistamines and decongestants may be separated efficaciously on a phenyl column.