Analytical Column Selection Guide
A complete decision path from sample properties to a specific grade. Includes a separation mode decision table, a stationary phase selectivity map, pore size versus molecular weight bands, van Deemter curves and the particle size trade-off, retention versus pH, detector constraints, a quick reference for nine application areas, pharmacopoeial correspondence and the link to preparative scale-up, with calculation methods and a source index.
1. Conditions to Define Before Selection SELECTION INPUTS
Selection is a constrained decision. Before asking "which column", the six conditions below should be established; where they are unclear, any selection can only be tentative.
| Condition | What needs to be established | Effect on selection |
|---|---|---|
| Sample properties | Molecular weight, polarity (logP), acid/base character (pKa), whether charged, whether it contains a chiral centre, solubility | Determines the separation mode and stationary phase chemistry |
| Purpose of the analysis | Assay / related substances / impurity identification / stability study / pre-preparative assessment | Determines the efficiency required, the sample load and the column length |
| Matrix complexity | Plasma, fermentation broth, food extract or purified intermediate | Determines whether a guard column is needed and how much contamination tolerance is required |
| Instrument conditions | System pressure limit, column oven range, detector type, system dead volume | Determines the minimum particle size and minimum column bore |
| Method status | In-house method / pharmacopoeial method / customer-specified method / to be filed for registration | Determines whether the column must be chosen by USP L-number and whether a factory test report is needed |
| What comes next | Whether it will be scaled to semi-preparative or preparative | Determines whether to choose a series that also offers the same phase in preparative grades |
2. Selecting the Separation Mode SEPARATION MODE
The separation mode is determined jointly by the sample's solubility, polarity and molecular size. The figure below lists the corresponding modes and common stationary phases by decision criterion. Most small-molecule drug analysis falls in the reversed-phase range, but polar compounds, ionic compounds and macromolecules each have a more suitable mode.
The same sample can sometimes be run in more than one mode. Relatively polar nucleosides, for example, can be run on HILIC, on a polar-embedded phase, or on an aqueous-stable reversed-phase column. The choice then usually rests on detector compatibility (the high organic content of HILIC favours MS response), consistency with an existing method, and equilibration time (HILIC generally equilibrates more slowly than reversed phase).
3. Reversed-Phase Stationary Phases & Selectivity STATIONARY PHASE
Once reversed phase is settled, the choice of stationary phase mainly affects two things:retention strengthandSelectivity. Retention strength determines whether components fall in a suitable retention factor range (generally k = 2–10), and selectivity determines whether adjacent components can be separated. Chain length (C18 / C8 / C4) mainly adjusts retention strength, while aromatic, fluorinated and polar-embedded phases are used to change selectivity.
| Stationary phase | Retention characteristics | When to Use This Service | Points to note |
|---|---|---|---|
| C18（ODS） | Strong retention, high carbon load | Most small-molecule drugs, impurity profiling, food and environmental analysis | Some products risk phase collapse under high aqueous conditions; choose an aqueous-stable grade |
| C8 | Retention about 1/2 to 2/3 that of C18 | Strongly hydrophobic compounds, shortening run time | Selectivity close to C18, so of limited use for improving resolution |
| C4 | Weak retention | Peptides, proteins, macromolecules | Retention is easily insufficient for small molecules |
| Phenyl / biphenyl | π–π interaction gives aromatic selectivity | Aromatic structural analogues, positional isomers | Selectivity weakens with acetonitrile-rich mobile phases; more pronounced in methanol systems |
| Pentafluorophenyl (PFP) | Dipole, π–π and steric effects together | Halogenated compounds, polar isomers, basic compounds | Selectivity is fairly sensitive to mobile phase composition; re-verify on method transfer |
| Polar-embedded / aqueous-stable | A hydration layer forms at the amide, carbamate or other polar group | 100% aqueous conditions, polar compounds, improved peak shape for basic compounds | Hydrophobic retention is slightly below a conventional C18 of the same carbon number |
| Cyano (CN) | Intermediate polarity, usable in both normal and reversed phase | Rapid screening, normal-phase separation | Batch reproducibility is generally lower than C18; keep this in view during method validation |
| Amino (NH₂) / diol | Predominantly polar interaction | Carbohydrates, normal-phase separation, HILIC mode | Amino phases can react with reducing sugars, aldehydes and ketones, giving a shorter life |
Phase classes and the grades available on this site
The table maps the phase classes above onto our three product lines:COSMOSIL is the Nacalai Tesque analytical column range, with a fairly complete set of special selectivity phases;HPLCONE is our own brand, built on SilicaOne custom-bonded silica, continuous from analytical to preparative and labelled with USP L-numbers;Daisogel is Osaka Soda bulk spherical silica, for self-packed columns and industrial preparative work.
| Phase class | COSMOSIL (analytical) | HPLCONE (analytical / preparative) | Daisogel (bulk silica) |
|---|---|---|---|
| General-purpose C18 | 5C18-MS-II (fully endcapped), 5C18-AR-II (partly endcapped, complementary selectivity) | 5C18A (300 m²/g surface area, high retention and high loading) | SP-120-ODS-RPS (17% carbon), SP-100-ODS-HP (24% carbon) |
| Acid- and base-stable C18 | — | 5C18C (pH 1.5–12, regenerable with NaOH) | SP-120-ODS-BIO (20% carbon, alkali regenerable) |
| Hydrophilic / aqueous-stable C18 | 5C18-PAQ | 5C18D (hydrophilic, for LC-MS), Ami C18 (amide polar-embedded) | SP-60-ODS-RPS (60 Å, 450 m²/g surface area) |
| C8 | 5C8-MS | C8 series | SP-120-C8-P、SP-200-C8-BIO |
| C4 / macromolecule reversed phase | 5C4-MS、5C18-AR-300、5C8-AR-300（300 Å） | — | SP-300-C4-BIO (3% carbon), SP-300-ODS-BIO (8% carbon) |
| Phenyl / aromatic selectivity | 5PE-MS (phenyl), πNAP (naphthylethyl), 5PYE (pyrenylethyl), PBr (pentabromophenyl), PFP (pentafluorophenyl) | PE phenyl (USP L11) | SP-120-C4Ph-HP (phenyl) |
| Shape selectivity | Cholester (cholesteryl), 5C22-AR-II (C22) | — | — |
| Cyano (CN) | 5CN-MS | — | — |
| Amino / carbohydrates | — | NH PLUS (polymer-based amino, USP L8), SUGAR-Ca / SUGAR-H | SP-120-APS-P (aminopropyl) |
| HILIC | HILIC | HILIC（USP L114 / L122） | — |
| Silica / normal phase | 5SL-II | SIL (USP L3, 320 m²/g surface area) | SP-120-P, SP-1000-P and other pore sizes |
| Chiral | CHiRAL 3A / 3B / 3C、5A / 5B / 5C | — | — |
4. Substrate, Bonding & Endcapping SILICA & BONDING
At the same "C18", differences in peak shape and column life between products usually come from the substrate and the bonding process rather than the carbon chain itself. The four parameters below are normally given in the product documentation and are worth comparing item by item.
| Parameter | Range | Effect |
|---|---|---|
| Silica purity | Ordinary silica / high-purity silica (metal impurities at ppm level) | Residual metals interact with chelating compounds, causing tailing and reduced response |
| Endcapping | Fully / partially / not endcapped | Residual silanols interact with basic compounds and cause tailing; non-endcapped products behave differently under high aqueous conditions |
| Bonding chemistry | Mono- / di- / trifunctional | Polyfunctional bonding gives better acid resistance; monofunctional bonding gives a more uniform layer |
| pH tolerance | Typically 2–8; base-stable grades reach 12; hybrid or polymer substrates are wider still | Determines the usable mobile phase pH range and cleaning options |
| Carbon load | About 7%–20% (varying with pore size and chain length) | Correlates with retention strength, but should not be compared directly across pore sizes |
| Maximum temperature | Typically 60 °C; some products 80 °C | A higher column temperature lowers viscosity and raises efficiency, but accelerates silica dissolution |
Working near the pH limits, hydrolysis of the silica substrate and loss of the bonded phase are among the main routes to shortened column life. Below pH 2 the bonded phase hydrolyses and is lost; above pH 8 the rate of silica skeleton dissolution rises. If the method must run at pH 9–12, choose a base-stable bonded silica, a hybrid substrate or a polymer substrate; lowering the column temperature also slows degradation.
5. Matching Pore Size to Molecular Weight PORE SIZE
Pore size determines whether the solute can move freely in and out of the pores. When molecular size approaches the pore size, mass transfer is impeded, showing up as band broadening and loss of efficiency. As a rule the pore size should be at least three times the solute's radius of gyration. The figure below gives the usual correspondence.
Pore size bands and surface areas available on this site
As pore size increases, surface area falls, and retention and sample load fall with it. The Daisogel bulk silica pore size bands and their parameters are given below as a reference for selecting by pore size; COSMOSIL and HPLCONE analytical columns are mostly in the 90–130 Å range, with a 300 Å grade for proteins and large peptides.
| Pore size | Surface area | Pore volume | ODS carbon load* | Suited to |
|---|---|---|---|---|
| 60 Å | 450 m²/g | 0.75 mL/g | 19%（RPS） | Small molecules and synthetic peptides where sample load matters |
| 100 Å | 320 – 450 m²/g | 0.9 – 1.1 mL/g | 17%（P）／24%（HP） | General-purpose reversed phase |
| 120 Å | 300 m²/g | 1.0 – 1.1 mL/g | 17%（RPS）／20%（BIO） | The workhorse band for general reversed phase |
| 200 Å | 200 m²/g | 1.1 mL/g | 12%（RPS）／15%（BIO） | Medium molecular weight peptides |
| 300 Å | 100 m²/g | 0.9 mL/g | 8%（BIO） | Proteins, large peptides, oligonucleotides |
| 1000 Å | 25 m²/g | 0.9 mL/g | — | Macromolecules, polymers |
| 2000 Å | 15 m²/g | 0.8 mL/g | — | Very large molecules |
Pore size selection in SEC follows different rules from reversed phase: SEC separates by pore size distribution, so the analyte must fall within the column's separation range (the linear part of the calibration curve) — bigger pores are not simply better. Samples with a wide molecular weight distribution can be run on columns of different pore size in series.
6 · Particle Size, Column Length and Backpressure GEOMETRY & EFFICIENCY
Efficiency rises as particle size falls, while backpressure rises with the inverse square of particle size. Choosing a particle size is really a balance betweenseparating power, run time and system pressure limit. The figure below shows plate height versus linear velocity (the van Deemter curve) for different particle sizes.
| Particle size | Usual column lengths | Typical backpressure (4.6 mm × 150 mm, 1.0 mL/min) | Suited to |
|---|---|---|---|
| 10 μm | 250 mm | About 30–60 bar | Routine detection, teaching, semi-preparative |
| 5 μm | 150 / 250 mm | About 80–150 bar | Covers most analytical work; commonly used in pharmacopoeial methods |
| 3 μm | 100 / 150 mm | About 200–350 bar | Shorter run times, complex impurity profiles |
| < 2 μm | 50 / 100 mm | Usually requires a system rated above 600 bar | UHPLC platform; system dead volume must be optimized to match |
It follows that doubling the column length doubles efficiency but raises resolution only about 1.4-fold, while doubling backpressure and run time as well. Going from 5 μm to 3 μm at the same column length raises efficiency about 1.7-fold and backpressure about 2.8-fold. So where the system's pressure rating allows, reducing particle size is usually more efficient than lengthening the column.
| Column ID | Usual flow rate | Relative sensitivity* | Suited to |
|---|---|---|---|
| 4.6 mm | 0.8 – 1.5 mL/min | 1.0 (reference) | Routine analysis; the usual format in pharmacopoeial methods |
| 3.0 mm | 0.4 – 0.7 mL/min | About 2.4 | Saves solvent, compatible with conventional HPLC |
| 2.1 mm | 0.2 – 0.4 mL/min | About 4.8 | First choice for LC-MS; requires a low dead volume system |
| 1.0 mm and below | < 0.1 mL/min | About 21 | Small samples; demanding on the system |
7 · Mobile Phase and pH MOBILE PHASE & pH
For ionizable compounds, mobile phase pH usually affects retention more than the organic proportion does. The figure below shows the general form of retention versus pH for acidic and basic compounds.
For stable retention, the mobile phase pH should generally sit more than two units away from the analyte's pKa, so that it exists in a single form. Within pKa ± 1, retention time is sensitive to pH variation and method robustness suffers.
| Buffer system | Effective pH range | MS compatible | Notes |
|---|---|---|---|
| Phosphoric acid / phosphate | 1.9–3.1；6.2–8.2 | No | Low UV cut-off, commonly used in pharmacopoeial methods; not usable with MS or ELSD |
| Formic acid / ammonium formate | 2.8–4.8 | Yes | Common in LC-MS, with good response in positive ion mode |
| Acetic acid / ammonium acetate | 3.8–5.8 | Yes | Fairly wide applicability, usable in both positive and negative ion mode |
| Ammonium bicarbonate | 9.2–10.2 | Yes | For MS methods under basic conditions; requires a base-stable column |
| Trifluoroacetic acid (TFA) | About 2.0 | Limited | Improves peptide peak shape but suppresses ESI response; quantitative methods must assess this |
As for organic modifiers, acetonitrile is a stronger eluent than methanol and less viscous, so it generates less backpressure; the hydrogen bonding of methanol offers different selectivity, most noticeably on phenyl columns. During method development the two can be screened as independent variables.
8. Detector-Imposed Constraints DETECTOR CONSTRAINTS
The detector determines which mobile phase components are unusable, and this constraint generally comes before the choice of column. Discovering late in method development that the mobile phase and detector are incompatible often means selecting a new column.
| Detector | Mobile phase restrictions | Column preference | Notes |
|---|---|---|---|
| UV / DAD | Must be transparent at the detection wavelength; acetonitrile has a lower cut-off than methanol | No particular restriction | At low wavelengths (< 210 nm), avoid carbonyl solvents and TFA |
| Fluorescence (FLD) | Avoid fluorescence quenchers | Matched to the derivatization scheme | Amino acids detected after OPA / FMOC derivatization, with higher sensitivity than UV |
| Refractive index (RI) | Isocratic only; column and ambient temperature must be stable | Dedicated carbohydrate columns, SEC columns | A gradient causes baseline drift and cannot be used |
| ELSD / CAD | The mobile phase must be entirely volatile; phosphate is prohibited | Reversed phase or HILIC | Response is non-linear with concentration; quantify with a logarithmic fit or a narrow calibration range |
| Mass spectrometry (MS) | Non-volatile salts prohibited; ion-pairing reagents suppress response | 2.1 mm bore columns, MS-compatible phases | Ammonium formate and ammonium acetate are the usual choices; the degree of suppression by TFA must be assessed |
| Conductivity / suppressed | Requires a matching eluent system | Dedicated ion chromatography columns | Analysis of inorganic anions and cations |
9 · Applications and Product Correspondence APPLICATION MATRIX
The table gives a usual starting point by sample class. The grades listed are current products on this site; the actual selection must still be checked against the conditions in Chapter 1.
| Sample class | Recommended starting point | Mobile phase system | Alternatives and notes |
|---|---|---|---|
| Peptides · short peptides | HPLCONE 5C18A (high loading) / 5C18C (acid- and base-stable); Daisogel SP-120-ODS-RPS for preparative work | 0.1% TFA or formic acid / acetonitrile gradient | For large peptides, switch to C4 or a 300 Å pore size; at preparative scale, watch the sample load |
| Related substances in drugs | COSMOSIL 5C18-MS-II or HPLCONE 5C18A, 5 μm × 150 mm | Buffer / acetonitrile gradient | For co-eluting components switch to phenyl or PFP; methods for filing must fix the brand and lot information |
| Carbohydrates · honey | HPLCONE SUGAR-Ca (calcium form) / SUGAR-H (hydrogen form) | Pure water or dilute sulfuric acid, isocratic | RI detection, column temperature 60–80 °C; the hydrogen form can also separate organic acids |
| Amino acids | HPLCONE 5C18D with pre-column derivatization (OPA / FMOC) | Buffer / acetonitrile gradient | Configuration can be analysed with a chiral derivatizing reagent, without a chiral column |
| Nucleotides · oligosaccharides | HPLCONE 5C18D, COSMOSIL 5C18-PAQ or a HILIC phase | High aqueous or high acetonitrile | A hydrophilic C18 retains stably in a fully aqueous mobile phase, which suits MS coupling |
| Polar metabolites (LC-MS) | COSMOSIL HILIC or HPLCONE HILIC (zwitterionic) | Acetonitrile / ammonium acetate gradient | Equilibration takes longer; equilibrate fully before starting a sequence |
| Aromatics · polycyclic aromatic hydrocarbons | COSMOSIL 5PYE / πNAP / PBr; HPLCONE PE phenyl for routine aromatics | Methanol / water or acetonitrile / water | π–π interaction gives selectivity complementary to C18 |
| Protein molecular weight distribution | Shodex aqueous SEC series (pore size chosen by separation range) | Phosphate buffer, isocratic | Keep conditions mild to preserve the native conformation; avoid non-specific adsorption on the substrate |
| Chiral drugs | COSMOSIL CHiRAL 3A–3C / 5A–5C; ChiralONE series for preparative work | Normal phase, reversed phase or polar organic mode | Screen the mobile phase mode first, then fix the phase; bonded types tolerate a wider solvent range |
COSMOSIL — special selectivity phases
COSMOSIL is made by Nacalai Tesque, with Microwants as distributor for mainland China. Beyond the usual C18 / C8 / C4, the range offers a number of aromatic and shape-selective phases, suited to structural analogues that C18 cannot resolve.
| Grade | Stationary phase | Mechanism | Typical use |
|---|---|---|---|
| 5C18-MS-II | ODS, fully endcapped | Hydrophobic | Routine analysis, good peak shape for basic compounds |
| 5C18-AR-II | ODS, partly endcapped | Hydrophobic + residual silanol interaction | Complementary selectivity to MS-II |
| 5C18-PAQ | Aqueous-stable ODS | Hydrophobic; no collapse at high aqueous content | Polar compounds, 100% aqueous conditions |
| 5C22-AR-II | C22 | Hydrophobic + shape selectivity | Long-chain and configurational isomers |
| Cholester | Cholesteryl | Shape selectivity | Fat-soluble vitamins, steroids |
| πNAP | Naphthylethyl | π–π | Aromatic positional isomers |
| 5PYE | Pyrenylethyl | π–π + dispersion forces | Polycyclic aromatics, fullerene-related separations |
| PBr | Pentabromophenyl | Hydrophobic + dispersion forces | Halogenated compounds, steroids |
| PFP | Pentafluorophenyl | π–π + dipole | Halogenated compounds, polar isomers |
| 5PE-MS | Phenyl | π–π | Aromatic compounds |
| 5C18-AR-300 / 5C8-AR-300 | 300 Å wide-pore ODS / C8 | Hydrophobic | Proteins and large peptides |
| CHiRAL 3A–3C / 5A–5C | Polysaccharide chiral phase | Chiral recognition | Enantiomer separation |
HPLCONE — our own brand, labelled with USP L-numbers
HPLCONE uses our own SilicaOne custom-bonded silica (total metal impurities < 10 ppm), with continuous analytical and preparative specifications and USP packing codes labelled on several grades, making column selection for pharmacopoeial methods straightforward.
| Grade | USP code | Characteristics | Suited to |
|---|---|---|---|
| 5C18A | L1 | 300 m²/g surface area, 12 nm pores, pH 2–10, usable in 100% aqueous | Peptides, antibiotics, natural products, balancing retention against sample load |
| 5C18C | L1 | High bonding density and full endcapping, pH 1.5–12, regenerable with NaOH | Small peptide separation, methods with demanding acid or base conditions |
| 5C18D | L1 | Hydrophilic bonding, no collapse in fully aqueous systems, no ion-pairing additive needed | Oligosaccharides, amino acids, nucleotides, organic acids, LC-MS coupling |
| Ami C18 | L60 / L1 | Amide polar-embedded, multi-mode interaction | Acids, amines, phenols and other compounds insufficiently retained on C18 |
| PE phenyl | L11 | π–π interaction | Aromatics and structural analogues |
| SIL | L3 | Spherical fully porous ultra-pure silica, 320 m²/g surface area | Normal-phase separation |
| NH PLUS | L8 | Polymer-based bonded amino, tolerant of alkaline cleaning | Carbohydrate quantification |
| HILIC | L114 / L122 | Zwitterionic hydrophilic phase | Polar compounds, metabolomics, glycopeptides |
| SUGAR-Ca / SUGAR-H | L19 / L17 | Ligand exchange, pure water mobile phase, column temperature 60–80 °C | Carbohydrates in honey and food; the hydrogen form also detects organic acids |
Daisogel — bulk silica for self-packing and scale-up
Daisogel is Osaka Soda spherical silica, with Microwants as China master distributor. Grades are named as "pore size – particle size – functional group – grade", with pore sizes of 60–2000 Å and particle sizes of 3–50 μm; the same phase runs continuously from analytical to industrial preparative particle sizes, which keeps selectivity constant on scale-up.
| Grade | Characteristics | Suited to |
|---|---|---|
| P | Conventional bonding, general purpose | Most routine separations |
| RPS | High bonding density, fairly complete endcapping | The workhorse for general reversed phase; SP-120-ODS-RPS at 17% carbon |
| HP | High carbon load | Where retention and sample load matter; SP-100-ODS-HP at 24% carbon |
| BP | Lower carbon load, slightly more polar | Gives selectivity different from RPS |
| BIO | Modified silica with a wider pH tolerance, regenerable with NaOH | Peptide and protein purification, processes requiring alkaline regeneration |
| PK | Flagship grade, with tighter batch consistency | Registered processes and long-term supply |
10. Pharmacopoeial Methods & Compliant Selection PHARMACOPOEIA
When running a pharmacopoeial method, the choice of column is constrained by the monograph. Pharmacopoeias specify a packing code rather than a brand, so products may be substituted within the same code, but system suitability must be demonstrated after any change.
| System | How it is specified | Permitted adjustment |
|---|---|---|
| USP | Specifies the packing class by L-number (L1 octadecylsilane bonded silica, L7 octyl, L11 phenyl, L3 silica, L10 cyano, L8 amino) | Under USP <621>, in isocratic methods the column length and particle size may be adjusted so long as L/dp stays within −25% to +50%; the limits for gradient methods are stricter |
| ChP (Chinese Pharmacopoeia) | General chapter 0512 sets the chromatographic conditions and system suitability requirements | Changes to the packing class and mobile phase composition must comply with the general chapter, and system suitability must be re-established |
| EP | Specified by packing description | Permitted adjustments are set out in general chapter 2.2.46 |
System suitability usually covers four items: theoretical plate number, tailing factor, resolution and repeatability. All four should be re-measured and recorded after a column change. For methods intended for regulatory filing, it is advisable to retain the brand, grade, lot number and factory test report of the column used.
11. Transition to Preparative Scale SCALE-UP
If the analytical method will later be scaled to semi-preparative or preparative work, the transition should be considered at the selection stage. The three points below, settled during the analytical phase, reduce rework on scale-up.
| Item | What to do at the analytical stage | Benefit on scale-up |
|---|---|---|
| Availability of the phase | Choose a phase series that also exists in 10 μm and 20 μm preparative grades | Selectivity is preserved on scale-up, with no need to redevelop the method |
| Scalability of the mobile phase | Avoid non-volatile salts such as phosphate that are difficult to recover | Lowers the cost of downstream desalting and solvent recovery |
| Loading conditions | Record the overload threshold (where injection amount and peak height depart from linearity) | Scale the loading in proportion to media mass, giving a sound basis for capacity estimates |
On availability of the phase, a given Daisogel pore size and functional group combination can be had at 3, 5, 10, 15, 20, 30, 40 and 50 μm, so selectivity can be confirmed at analytical scale with a small particle and the same bonded phase carried to preparative scale at a larger particle, which avoids re-screening phases on scale-up. HPLCONE analytical and preparative columns use the same SilicaOne substrate, with specifications extending from 4.6 mm bore to 20 mm and custom sizes.
Packing and acceptance requirements after scale-up are covered in Analytical Column Packing、DAC Packing Support and DAC Packing Problem Diagnosis。
12. Selection Self-Check List CHECKLIST
| No. | Check | Action if it fails |
|---|---|---|
| 1 | The separation mode is consistent with the sample's polarity, molecular weight and charge state | Return to Chapter 2 and re-decide |
| 2 | k for the target components falls in the range 2–10 | Adjust the organic proportion; if it is still outside, change chain length |
| 3 | Resolution for the critical pair is ≥ 1.5 | Change the stationary phase selectivity first, and adjust the gradient second |
| 4 | Mobile phase pH is more than two units away from the analyte's pKa | Adjust the pH, or move to ion pairing or HILIC |
| 5 | Mobile phase pH is within the chosen column's tolerance | Switch to a base-stable, hybrid or polymer substrate |
| 6 | Pore size matches the sample's molecular weight | Adjust the pore size per Chapter 5 |
| 7 | Expected backpressure does not exceed 80% of the system limit | Increase particle size, shorten the column, or reduce the flow rate |
| 8 | Mobile phase is compatible with the detector | Change the buffer system, and reselect the phase if necessary |
| 9 | A guard column is fitted for complex matrices | Add a guard cartridge of the same phase |
| 10 | For a pharmacopoeial method, the L-number and system suitability requirements have been checked | Consult Pharmacopoeia and re-measure the four criteria |
| 11 | If scale-up is intended, preparative grades of the same phase are confirmed available | Change to a phase series that has preparative grades |
Appendix A Calculation Methods and Assumptions METHODS & ASSUMPTIONS
The equations used on this page are set out below for checking and recalculation. Symbols: tR retention time, t0 dead time, W1/2 width at half height, L column length, dp particle size, dc column bore, ε total column porosity (taken as 0.65 for estimation), F flow rate.
The backpressure ranges given are estimated for an acetonitrile/water mobile phase (about 50:50, 25 °C, viscosity about 0.8 mPa·s) and vary with mobile phase composition, column temperature and medium batch; they are for initial selection, not for acceptance. Relative sensitivity is calculated for equal injected amounts with peak volume inversely proportional to column cross-sectional area, without allowing for system broadening or the injection volume limit, so measured values are generally lower than calculated.
The pore size to molecular weight ranges are general empirical values (inferred for this page; no statistical source); the coordinates in the stationary phase map indicate relative relationships and do not represent measured parameters for any product.
Appendix B Source Index REFERENCES
This page is compiled from the public sources and existing site data below. Pharmacopoeial text follows the current edition; please check the latest version when citing.
| Pharmacopoeias and regulations | ||
| [1] | USP general chapter <621> Chromatography, including the L-number packing classification and permitted method adjustments | usp.org |
| [2] | USP general chapter <1225> Validation of Compendial Procedures | usp.org |
| [3] | Pharmacopoeia of the People's Republic of China, 2020 edition, Part IV, general chapter 0512, High Performance Liquid Chromatography | chp.org.cn |
| [4] | European Pharmacopoeia 2.2.29 Liquid chromatography; 2.2.46 Chromatographic separation techniques | edqm.eu |
| [5] | ICH Q2(R2) Validation of Analytical Procedures | ich.org |
| [6] | ICH Q3A(R2) / Q3B(R2) Impurities in new drug substances and new drug products | ich.org |
| [7] | ICH Q14 Analytical Procedure Development | ich.org |
| Monographs and methodology | ||
| [8] | Snyder L.R., Kirkland J.J., Dolan J.W. Introduction to Modern Liquid Chromatography, 3rd ed., Wiley | — |
| [9] | Snyder L.R., Kirkland J.J., Glajch J.L. Practical HPLC Method Development, 2nd ed., Wiley | — |
| [10] | Neue U.D. HPLC Columns: Theory, Technology, and Practice, Wiley-VCH | — |
| [11] | Guiochon G. et al. Fundamentals of Preparative and Nonlinear Chromatography, 2nd ed., Academic Press | — |
| [12] | van Deemter J.J., Zuiderweg F.J., Klinkenberg A. Chem. Eng. Sci., 1956, 5, 271 — plate height and linear velocity | — |
| [13] | Knox J.H. J. Chromatogr. Sci. — reduced parameters and packing quality criteria | — |
| [14] | IUPAC Recommendations, Nomenclature for Chromatography (Pure Appl. Chem., 1993, 65, 819) | iupac.org |
| Product and site material | ||
| [15] | COSMOSIL column technical documentation (Nacalai Tesque) | nacalai.co.jp |
| [16] | Daisogel chromatographic silica product documentation (Osaka Soda DAISO) | daiso-chem.co.jp |
| [17] | Pages on this site:Pharmacopoeia section (USP L-number reference)、FAQ、Troubleshooting Manual、Calculators | microwants.com |
| [18] | Pages on this site:Analytical Column Packing、DAC Packing Support、DAC Packing Problem Diagnosis | microwants.com |