—
Analytical conditions are determined by the
compound, not by the
instrument —
Prerequisites for Method
Development
When performing HPLC analysis,
the first requirement is access to an HPLC system that can be used
freely. Method development is not
a task that can be completed in a short time. Even for simple
compounds, at least one full day is usually required. For
structurally complex compounds or multicomponent separations, it is
not uncommon for method development to take several weeks or even
months.
If the developed method is
intended to be transferred and used on different instruments or at
different facilities, the use of a
high-pressure gradient
system is strongly recommended. Low-pressure gradient
systems are highly instrument-dependent and often suffer from poor
reproducibility.
Information Gathering:
Analysis Starts with Searching
Once access to an HPLC system
has been secured, the next step is to collect information about the
compound of interest.
Literature searches and web
searches using the compound name
Use of analytical databases
provided by HPLC instrument manufacturers and column
manufacturers
For example, the Imtakt Column
Application Database allows users to search analytical conditions by
compound name or compound class:
https://www.imtakt.com/DB/
From these sources, existing
analytical conditions, separation modes, and detection methods can
be identified.
Importance of Structural
Information
If no analytical information is
available for a compound, it is essential to investigate its
molecular structure. Setting
analytical conditions without knowing the structure of the compound
has an extremely low probability of success, both empirically and
scientifically.
All factors governing HPLC
separation are based on molecular structure, including:
Polarity and hydrophobicity
Ionic character (cationic,
anionic, zwitterionic)
Properties of ionizable
groups (pKa, isoelectric point, pI)
Types of functional groups
(saturated and unsaturated bonds, amides, esters, etc.)
Physicochemical properties
relevant to detection (e.g., UV absorbance, MS response)
It is virtually impossible to
determine appropriate analytical conditions based solely on the
compound name. Even if an
existing method is found, blindly following it without understanding
why the conditions work
will never lead to true method development.
Selection of Separation Mode
and Stationary Phase
In the initial stage of method
development, it is common practice to first evaluate whether the
compound can be retained under reversed-phase conditions (ODS
columns such as
Cadenza
CD-C18 or
Unison
UK-C18).
If sufficient hydrophobic
retention cannot be achieved, alternative separation modes should be
considered, such as:
When selecting a column, it is
highly effective to consult the column selection guides provided by
manufacturers and refer to applications involving compounds with
similar structures or separation requirements.
Column internal diameter should
be selected according to the detector and sample amount.
For LC-MS and trace-level
analyses, a 3 mm
internal diameter is recommended.
Compared with 2 mm columns, 3 mm columns provide a better
balance between theoretical plate number and operational
robustness, and are well suited for gradient analysis.
Column Length
In the early stages of
method development, relatively short columns (50–75
mm) are recommended, as they allow rapid evaluation of multiple
conditions.
For challenging separations
such as isomer separation, it may be necessary to begin with
longer columns (150 mm) to achieve sufficient resolution.
In multicomponent
separations, conditions may first be optimized on a 100 mm
column, followed by high-resolution separation using a 250 mm
column with long analysis times (e.g., 60 minutes).
Selection of Detector
The detector should be selected
based on the molecular structure of the analyte and the analytical
objective.
UV-VIS detectors
Suitable for aromatic compounds, conjugated systems, and
compounds with UV-absorbing functional groups.
MS detectors
Effective for high-sensitivity analysis, compound
identification, and analysis in complex matrices.
Detector selection also affects
mobile phase composition (e.g., volatility and salt concentration).
When considering not only the detector but also column lifetime, the
use of volatile acids, salts, and buffers—such as acetic
acid/ammonium acetate or formic acid/ammonium formate—is strongly
recommended.
Basic Strategy for Mobile
Phase Design
In reversed-phase HPLC, the
mobile phase is typically based on the following combination:
At the initial stage of method
development, isocratic
conditions should not be set arbitrarily.
Instead, retention behavior should first be evaluated under
gradient conditions,
and isocratic conditions should be established only if necessary,
based on the results obtained.
When isocratic conditions are
finalized, a single,
premixed mobile phase must be prepared.
Mobile phase preparation by on-line pump mixing may result in
insufficient molecular-level homogeneity due to mixer efficiency and
flow-path design, which can raise concerns regarding
reproducibility.
Another important consideration
in isocratic analysis is the
strength of intermolecular
interactions.
Hydrophobic interactions are
relatively weak and may allow successful isocratic operation.
In contrast, electrostatic
and ionic interactions are much stronger, and even under
isocratic conditions, changes in retention time and peak shape are
likely to occur when the column is replaced. This is due to
differences in effective surface area and functional group density
of the stationary phase.
Just as elution in ion-exchange
chromatography is controlled by changes in ionic strength,
attempting isocratic
analysis of compounds possessing polar or ionic functional groups
based on electrostatic or ionic interactions is not recommended at
all from the standpoint of reproducibility.
In such cases, gradient
elution should be employed.
In practice, many complaints
regarding “poor column-to-column reproducibility” arise from mobile
phase designs that fail to adequately consider the interaction
characteristics of the analytes.
The base materials used for
packed stationary phases are polymeric in nature, and
it is impossible with
current scientific technology to reproduce molecular weight and
structure with accuracy down to 1 Da.
Therefore, especially under conditions that rely heavily on strong
intermolecular interactions, it is essential to recognize that
column-to-column variability is more likely to become apparent.
For compounds with ionizable
functional groups, the following parameters strongly influence
separation behavior:
Mobile phase pH
Ionic strength
Type of counter-ion
pH adjustment is not merely a
matter of numerical control but an operation that governs chemical
equilibria. From the standpoint
of reproducibility, preparation methods that do not rely on pH
meters are often preferred.
When transferring a validated
method to another facility, reproducibility may be poor with
low-pressure gradient systems.
Therefore, high-pressure gradient systems are recommended when
method robustness and transferability are important.
Once peaks have been observed,
analytical conditions should be optimized while evaluating:
Peak shape
Retention time
Resolution
If necessary, the following
parameters should be reconsidered:
Type and ratio of organic
solvent
pH
Ionic strength
For quantitative analysis,
additional evaluations are required, including:
Calibration curves
Detection sensitivity
Linearity and
reproducibility
Injection Sample Solution
Ideally, the sample should
dissolve completely in the mobile phase, but this is not always the
case. Poor dispersion of the
analyte in the sample solution can cause peak distortion such as
leading.
The analyte in the injected
sample must be completely dispersed at the molecular level.
This is because HPLC separation is based on molecular-level
interactions between individual analyte molecules and stationary
phase molecules.
After establishing a method,
repeated analyses should be performed to confirm reproducibility.
If variations in retention time, peak area, pressure, or peak shape
are observed, the method should be considered non-robust.
In such cases, it is necessary
to re-evaluate the entire procedure, including sample preparation
and mobile phase composition (especially ionic strength).
Unstable samples may exhibit changes in peak height or the
appearance of impurities after repeated analyses. For such samples,
stabilization strategies or fresh sample preparation are required.
Some users hope to analyze all
compounds using a single ODS column, but this is impossible.
Molecular interactions between the compound and the stationary phase
depend on molecular properties, and appropriate stationary phases
must be selected accordingly. Molecular structure—particularly ionic
character—is essential information.
An analytical method that
succeeds by chance without consideration of molecular structure will
never possess reproducibility or applicability.
