Dissolution testing

From Wikipedia, the free encyclopedia

In the pharmaceutical industry, drug dissolution testing is routinely used to provide critical in
vitro drug release information for both quality control purposes, i.e., to assess batch-to-batch
consistency of solid oral dosage forms such as tablets, and drug development, i.e., to predict in
vivo drug release profiles.[1]

In vitro drug dissolution data generated from dissolution testing experiments can be related to in
vivo pharmacokinetic data by means of in vitro-in vivo correlations (IVIVC). A well established
predictive IVIVC model can be very helpful for drug formulation design and post-approval
manufacturing changes.[2]
The main objective of developing and evaluating an IVIVC is to establish the dissolution test as a
surrogate for human studies, as stated by the Food and Drug Administration(FDA). Analytical data
from drug dissolution testing are sufficient in many cases to establish safety and efficacy of a drug
product without in vivo tests, following minor formulation and manufacturing changes (Qureshi and
Shabnam, 2001). Thus, the dissolution testing which is conducted in dissolution apparatus must be
able to provide accurate andreproducible results.
Several dissolution apparatuses exist. In United States Pharmacopeia (USP) General Chapter
<711> Dissolution, there are four dissolution apparatuses standardized and specified. [3] They are:
USP Dissolution Apparatus 1 - Basket (37C)
USP Dissolution Apparatus 2 - Paddle (37C)
USP Dissolution Apparatus 3 - Reciprocating Cylinder (37C)
USP Dissolution Apparatus 4 - Flow-Through Cell (37C)
USP Dissolution Apparatus 2 is the most widely used apparatus among these four.
The performances of dissolution apparatuses are highly dependent on hydrodynamics due to the
nature of dissolution testing. The designs of the dissolution apparatuses and the ways of operating
dissolution apparatuses have huge impacts on the hydrodynamics, thus the performances.
Hydrodynamic studies in dissolution apparatuses were carried out by researchers over the past few
years with both experimental methods and numerical modeling such as Computational Fluid
Dynamics (CFD). The main target was USP Dissolution Apparatus 2.[1][4][5][6][7][8][9][10] The reason is that
many researchers suspect that USP Dissolution Apparatus 2 provides inconsistent and sometimes
faulty data.[11][12][13][14][15][16][17] The hydrodynamic studies of USP Dissolution Apparatus 2 mentioned above
clearly showed that it does have intrinsic hydrodynamic issues which could result in problems. In
2005, Professor Piero Armenante from New Jersey Institute of Technology (NJIT) and Professor
Fernando Muzzio from Rutgers University submitted a technical report to the FDA.[18] In this technical
report, the intrinsic hydrodynamic issues with USP Dissolution Apparatus 2 based on the research
findings of Armenante's group and Muzzio's group were discussed.
High-performance liquid
chromatography(HPLC)
From Wikipedia, the free encyclopedia

High-performance liquid chromatography

An HPLC. From left to right: A pumping device generating a gradient

of two different solvents- a steel-enforced column and a detector for

measuring the absorbance.

Acronym HPLC

Classification Chromatography

Analytes organic molecules

biomolecules

ions

polymers

Other techniques

Related Chromatography

Aqueous normal-phase chromatography

Hydrophilic Interaction Chromatography

Ion exchange chromatography

Size exclusion chromatography

Micellar liquid chromatography

Hyphenated Liquid chromatography-mass spectrometry

A modern self-contained HPLC.

Schematic representation of an HPLC unit. (1) Solvent reservoirs, (2) Solvent degasser, (3) Gradient valve, (4)
Mixing vessel for delivery of the mobile phase, (5) High-pressure pump, (6) Switching valve in "inject position",
(6') Switching valve in "load position", (7) Sample injection loop, (8) Pre-column (guard column), (9) Analytical
column, (10) Detector (i.e. IR, UV), (11) Data acquisition, (12) Waste or fraction collector.

High-performance liquid chromatography (HPLC; formerly referred to as high-pressure liquid

chromatography), is a technique in analytical chemistry used to separate, identify, and quantify
each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the
sample mixture through a column filled with a solid adsorbent material. Each component in the
sample interacts slightly differently with the adsorbent material, causing different flow rates for the
different components and leading to the separation of the components as they flow out the column.
HPLC has been used for manufacturing (e.g. during the production process of pharmaceutical and
biological products), legal (e.g. detecting performance enhancement drugs in urine), research (e.g.
separating the components of a complex biological sample, or of similar synthetic chemicals from
each other), and medical (e.g. detecting vitamin D levels in blood serum) purposes. [1]
Chromatography can be described as a mass transfer process involving adsorption. HPLC relies on
pumps to pass a pressurized liquid and a sample mixture through a column filled with adsorbent,
leading to the separation of the sample components. The active component of the column, the
adsorbent, is typically a granular material made of solid particles (e.g. silica, polymers, etc.), 250
micrometers in size. The components of the sample mixture are separated from each other due to
their different degrees of interaction with the absorbent particles. The pressurized liquid is typically a
mixture of solvents (e.g. water, acetonitrile and/or methanol) and is referred to as a "mobile phase".
Its composition and temperature play a major role in the separation process by influencing the
interactions taking place between sample components and adsorbent. These interactions are
physical in nature, such as hydrophobic (dispersive), dipoledipole and ionic, most often a
combination.
HPLC is distinguished from traditional ("low pressure") liquid chromatography because operational
pressures are significantly higher (50350 bar), while ordinary liquid chromatography typically relies
on the force of gravity to pass the mobile phase through the column. Due to the small sample
amount separated in analytical HPLC, typical column dimensions are 2.14.6 mm diameter, and 30
250 mm length. Also HPLC columns are made with smaller sorbent particles (250 micrometer in
average particle size). This gives HPLC superior resolving power (the ability to distinguish between
compounds) when separating mixtures, which makes it a popular chromatographic technique.
The schematic of an HPLC instrument typically includes a sampler, pumps, and a detector. The
sampler brings the sample mixture into the mobile phase stream which carries it into the column.
The pumps deliver the desired flow and composition of the mobile phase through the column. The
detector generates a signal proportional to the amount of sample component emerging from the
column, hence allowing for quantitative analysis of the sample components. A
digital microprocessor and user software control the HPLC instrument and provide data analysis.
Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in
ratios changing in time, generating a composition gradient in the mobile phase. Various detectors
are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry. Most
HPLC instruments also have a column oven that allows for adjusting the temperature at which the
separation is performed.

Operation[edit]

The sample mixture to be separated and analyzed is introduced, in a discrete small volume (typically
microliters), into the stream of mobile phase percolating through the column. The components of the
sample move through the column at different velocities, which are a function of specific physical
interactions with the adsorbent (also called stationary phase). The velocity of each component
depends on its chemical nature, on the nature of the stationary phase (column) and on the
composition of the mobile phase. The time at which a specific analyte elutes (emerges from the
column) is called its retention time. The retention time measured under particular conditions is
considered an identifying characteristic of a given analyte.

Many different types of columns are available, filled with adsorbents varying in particle size, and in
the nature of their surface ("surface chemistry"). The use of smaller particle size packing materials
requires the use of higher operational pressure ("backpressure") and typically improves
chromatographic resolution (i.e. the degree of separation between consecutive analytes emerging
from the column). In terms of surface chemistry, sorbent particles may be hydrophobic or polar in
nature.
Common mobile phases used include any miscible combination of water with various organic
solvents (the most common are acetonitrile and methanol). Some HPLC techniques use water-free
mobile phases (see Normal-phase chromatography below). The aqueous component of the mobile
phase may contain acids (such as formic, phosphoric ortrifluoroacetic acid) or salts to assist in the
separation of the sample components. The composition of the mobile phase may be kept constant
("isocratic elution mode") or varied ("gradient elution mode") during the chromatographic analysis.
Isocratic elution is typically effective in the separation of sample components that are not very
different in their affinity for the stationary phase. In gradient elution the composition of the mobile
phase is varied typically from low to high eluting strength. The eluting strength of the mobile phase is
reflected by analyte retention times with high eluting strength producing fast elution (=short retention
times). A typical gradient profile in reversed phase chromatography might start at 5% acetonitrile (in
water or aqueous buffer) and progress linearly to 95% acetonitrile over 525 minutes. Periods of
constant mobile phase composition may be part of any gradient profile. For example, the mobile
phase composition may be kept constant at 5% acetonitrile for 13 min, followed by a linear change
up to 95% acetonitrile.

A rotary fraction collector collecting HPLC output. The system is being used to isolate a fraction
containing Complex I from E. coli plasma membranes. About 50 litres of bacteria were needed to isolate this
amount.[2]

The chosen composition of the mobile phase (also called eluent) depends on the intensity of
interactions between various sample components ("analytes") and stationary phase (e.g.
hydrophobic interactions in reversed-phase HPLC). Depending on their affinity for the stationary and
mobile phases analytes partition between the two during the separation process taking place in the
column. This partitioning process is similar to that which occurs during a liquidliquid extraction but is
continuous, not step-wise. In this example, using a water/acetonitrile gradient, more hydrophobic
components will elute (come off the column) late, once the mobile phase gets more concentrated in
acetonitrile (i.e. in a mobile phase of higher eluting strength).
The choice of mobile phase components, additives (such as salts or acids) and gradient conditions
depends on the nature of the column and sample components. Often a series of trial runs is
performed with the sample in order to find the HPLC method which gives adequate separation.

pH meter
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A pH meter

A pH Meter is a scientific instrument that measures the hydrogen-ion concentration (or pH) in a
solution, indicating its acidity oralkalinity.[1] The pH meter measures the difference in electrical
potential between a pH electrode and a reference electrode. It usually has a glass electrode plus
a calomel reference electrode, or a combination electrode.[2] In addition to measuring the pH of
liquids, a special probe is sometimes used to measure the pH of semi-solid substances.

Uses[edit]

Knowledge of pH to greater or lesser accuracy is useful or critical in a great many situations,

including of course chemical laboratory work. pH meters of various types and quality can be used
for soil measurements in agriculture; water quality for water supply systems, swimming pools,
etc.; brewing, industrially or domestically; healthcare, to ensure that solutions are safe when applied
to patients or lethal as sterilants and disinfectants; and many other applications.
Circuit and operation[edit]

Basic potentiometric pH meters simply measure the voltage between two electrodes and display the
result converted into the corresponding pH value. They comprise a simple electronic amplifier and a
pair of probes, or a combination probe, and some form of display calibrated in pH. The probe is the
key part: it is a rod-like structure usually made of glass, with a bulb containing the sensor at the
bottom. Frequent calibration with solutions of known pH, perhaps before each use, ensures the best
accuracy.[3] To measure the pH of a solution, the probe is dipped into it.

Probe care and cleaning[edit]

Probes need to be kept clean of contamination as far as possible, and not touched by hand. Probes
are best kept moist with a medium appropriate for the particular probe (distilled water, which can
encourage diffusion out of the electrode, is undesirable) when not in use. [3][4] If the bulb becomes
contaminated with use it can be cleaned in the manner recommended by the manufacturer; a quick
rinse in distilled water immediately after use, blotted (not wiped) off may be sufficient. [3] One maker of
laboratory-grade equipment gives different cleaning instructions for general cleaning (15' soak in a
solution of bleach and detergent), salt (hydrochloric acid solution followed by sodium hydroxide and
water), grease (detergent or methanol), clogged reference junction (KCl solution), protein deposits
(pepsin and HCl, 1% solution), and air bubbles.[4][5]

Calibration and use[edit]

For very precise work the pH meter should be calibrated before each measurement. For normal use
calibration should be performed at the beginning of each day. The reason for this is that the glass
electrode does not give a reproducible e.m.f. over longer periods of time

Calibration should be performed with at least two standard buffer solutions that span the range of pH
values to be measured. For general purposes buffers at pH 4.00 and pH 10.00 are acceptable. The
pH meter has one control (calibrate) to set the meter reading equal to the value of the first standard
buffer and a second control which is used to adjust the meter reading to the value of the second
buffer. A third control allows the temperature to be set. Standard buffer sachets, which can be
obtained from a variety of suppliers, usually state how the buffer value changes with temperature.
For more precise measurements, a three buffer solution calibration is preferred. As pH 7 is
essentially, a "zero point" calibration (akin to zeroing or taring a scale or balance), calibrating at pH 7
first, calibrating at the pH closest to the point of interest (e.g. either 4 or 10) second and checking the
third point will provide a more linear accuracy to what is essentially a non-linear problem. Some
meters will allow a three-point calibration and that is the preferred scheme for the most accurate
work. Higher quality meters will have a provision to account for temperature coefficient correction,
and high-end pH probes have temperature probes built in. The calibration process correlates the
voltage produced by the probe (approximately 0.06 volts per pH unit) with the pH scale. After each
single measurement, the probe is rinsed with distilled water or deionized water to remove any traces
of the solution being measured, blotted with a scientific wipe to absorb any remaining water which
could dilute the sample and thus alter the reading, and then quickly immersed in a solution suitable
for storage of the particular probe type.[6]