The KISS principle is the preferred approach during method development. Simplicity should be a key goal while designing the method development strategy and unnecessary complexity should be avoided. For example, a gradient is not necessary, when analytes can be easily separated under isocratic conditions. Similarly, a ternary gradient is not the best choice, when a binary seems to be efficient. The separation of target analytes in HPLC mixtures relies on the following properties: charge, hydrophobicity, affinity, solubility, and molecular weight.
Partition chromatography, based on partitioning of analytes between two liquid phases. Ionic interactions depend on charge, ion size, and polarization. The pH also affects the separation. Size exclusion chromatography, also known as gel permeation or gel filtration chromatography, based on molecular size.
According to their size, some molecules are included in the pores of the stationary phase and thus they are retained, while some are excluded, so they pass through the column unretained. In this case, the pore size is critical and affects the retention and as a result the retention time. Affinity chromatography in which analytes enzymes, antibodies, etc. Before delving deeper into the various aspects of the method development, it is necessary to describe briefly some fundamental terms 7.
Usually, uracil or nitrite and nitrate salts can be used in reversed phase HPLC for the determination of t 0.
Any chromatographer wishes to obtain perfectly symmetric peaks, which can be more accurately quantified, but in the real world of chromatography, most peaks are asymmetric to some degree. Peak tailing factor T f and peak asymmetry factor A s are two terms that are used to describe the same phenomenon, and they result in slightly different numeric values due to the different calculation approach. In Equation 6 , peak width is measured at the baseline, whereas in 7 it is measured at the half height of the peak. The comparison of the performance of different chromatographic columns is made by the H versus u plots, known as the van Deemter plots.
The A , B , and C terms of the van Deemter equation are given by 12 — 14 relationships. Resolution is governed by different physicochemical phenomena. It is up to the chromatographer to obtain the desired resolution. Resolution can be influenced by changing one of the three parameters, selectivity, efficiency, and retention, as described by the Purnell equation Equation 8 , which determines the method development strategy. As expected by the square root sign, efficiency N has a relatively smaller effect on resolution, but it can significantly influence run time.
Resolution is improved as the peaks become narrower. While choosing the right packing material of the analytical column, the most common choice is the almost universal solution of octadecylsilane C 18 or ODS , which is very nonpolar. In this case, retention is based on London dispersion interactions with hydrophobic compounds. When considering the influence of the physical properties of the packing material, it is also important to take into account the influence of the column size, the particle size and shape, the surface area, the pore size, the carbon load, the bonding type, and the base material.
Column dimensions : Refer to the length of column and the internal diameter of the packing bed within the column. Narrow columns produce narrower and taller peaks and a lower limit of detection.
Particle shape and size : Chromatographic packing materials may be spherical particles or irregular in shape. Spherical particles offer reduced back pressures. Particle size : Refers to the average diameter of the particles. Although the manufacturers give a nominal size, this is typically the average size and some bigger or smaller particles are also included. Smaller particles allow less diffusion of analytes and thus they result in narrower and sharper peaks. However, smaller particles cause higher back pressures. Pore size : it refers to the average size of the pores in porous packing materials.
Larger pores allow larger solute molecules to be retained longer through maximum exposure to the surface area of the particles. The retention factor k is increased by decreasing the percentage of organic modifier in the mobile phase. The pH of mobile phase also affects k , in the case of weak organic acids and amines, depending on their p K a value. The solvent type e. A change in the percentage of organic modifier, the application of gradient elution, the type of gradient e. The Snyder triangle helps in finding the optimum mobile phase. The idea behind the triangle is that solvents in the same group will provide a comparable chromatographic selectivity.
Therefore, switching from one solvent to another within the same group would not yield a spectacular change in selectivity as expected by switching to a solvent in a group with different characteristics, the same way as it happens by switching from group I to group VII, for example. Snyder's triangle helps the chromatographer to choose wisely from among a selection of various solvents.
For example, if methanol in the mobile phase is ineffective, a similar result is expected from the use of another alcohol such as ethanol or propanol. Choosing a solvent from an entirely different part of the triangle is more likely to yield the desired separation. On the other hand, larger alcohols, such as propanol, tend to be less denaturing to biomolecules than methanol; so, it may be the solvent of choice in some cases. Controlling the pH can also play a significant role in changing the selectivity, when the analytes are weak acids or bases.
Ionized forms are strongly retained in ion chromatography but are retained less in reversed phase, while nonionized forms exhibit the opposite behavior. Buffer type and buffer concentration are among the factors that need to be taken into consideration during method development. Optimization process can also be performed by the prediction of elution times, t R , of the analytes under examination using mathematical models in an attempt to minimize both the solvent consumption and the trial time Temperature is a significant factor that is often neglected or underestimated in liquid chromatography because it is erroneously related to gas chromatography GC.
What are the Differences between GC and HPLC?
However, it can also be powerful in resolution control and can be a critical parameter in HPLC, but in a narrower range and to a lesser extent than in GC. Resistance in mass transfer the C term in van Deemter equation is significantly reduced in elevated temperatures. In nearly all separations, an increase in temperature will also cause a decrease in retention. Moreover, a decreased solvent viscosity at elevated temperatures leads to lower back pressure. This allows the use of higher flow rates using standard equipment. Since high temperature leads to a flatter van Deemter curve, it enables the use of higher flow rates without sacrificing efficiency and thus optimizing the resolution Figure 6.
However, disadvantages of elevated temperatures in HPLC should also be mentioned. For example, there are inconveniences related to instrumentation or type of columns. Undoubtedly, more research in this field is required The minimum standards of reporting checklist. Electronic supplementary material. Lei Sun, Email: moc. Hong-yu Jin, Email: nc. Run-tao Tian, Email: moc. Ming-juan Wang, Email: nc. Li-na Liu, Email: moc. Liu-ping Ye, Email: moc. Tian-tian Zuo, Email: moc.
Shuang-cheng Ma, Email: nc. National Center for Biotechnology Information , U. Journal List Chin Med v. Chin Med. Published online Jun Run-tao Tian 3 Chemmind Technologies Co.
- High-Performance Liquid Chromatography (HPLC) | Protocol?
- La Vecchia dellAceto: 5 (Vento della Storia) (Italian Edition).
- Chapter 13 Validation of Analytical Methods Based on Chromatographic Techniques: An Overview.
- 7 QUALITY OF ANALYTICAL PROCEDURES!
- Me Father Was A Hero And Me Mother Is A Saint Book.
Author information Article notes Copyright and License information Disclaimer. Corresponding author.
Materials and Methods
Received Feb 20; Accepted May This article has been cited by other articles in PMC. Associated Data Data Availability Statement All data generated or analysed during this study are included in this published article [and its supplementary information files]. Abstract Background Analysis of related substances in pharmaceutical chemicals and multi-components in traditional Chinese medicines needs bulk of reference substances to identify the chromatographic peaks accurately.
Results The method was validated in two medicines on 30 columns. Electronic supplementary material The online version of this article doi Background Multi-components analysis is an effective strategy for quality control of traditional Chinese medicines TCMs , which have complex chemical profiles. Methods The Minimum Standards of Reporting Checklist contains details of the experimental design, and statistics, and resources used in this study Additional file 3. Open in a separate window.
HPLC–Tandem Mass Spectrometric Method to Characterize Resveratrol Metabolism in Humans
Preparation of sample solution Psoraleae [ 26 ]: weigh 0. Chromatographic conditions Psoraleae [ 26 ]: mobile phase A was water and mobile phase B was methanol. Minimum number of columns for St R calculation Theoretically, t R on any column can be used as reference value for linear fitting. Prediction results of different number of columns for S tR calculation. Procedure of two points prediction For RR method, only one compound was chosen as reference compound reference substance required , and RR of all other compounds were used as reference value for calculating t R pre.
Procedure of multiple points regression After assignment of the peaks of analytes in the sample solution by prediction of two points regression, the t R mea of those peaks should be validated by multiple points regression. Sequential matching rule If the t R of two peaks are too close, e. Exclusion of column and compound by linear fitting Nonlinear shift of t R for a compound on different columns could be caused either by different column packing materials and use of other packing techniques, or by the different compound structure.
Selection of two reference compounds Ideally there should be no difference in selecting any of the two compounds as reference compounds. Acknowledgements We would like to thank Ms. Competing interests The authors declare that they have no competing interests. Availability of data and materials All data generated or analysed during this study are included in this published article [and its supplementary information files]. Additional file Additional file 1.
Footnotes Electronic supplementary material The online version of this article doi Contributor Information Lei Sun, Email: moc. References 1. One single standard substance for the determination of multiple anthraquinone derivatives in rhubarb using high-performance liquid chromatography-diode array detection. J Chromatogr A.
Ruggedness and robustness of conversion factors in method of simultaneous determination of multi-components with single reference standard. A single, multi-faceted, enhanced strategy to quantify the chromatographically diverse constituents in the roots of Euphorbia kansui. J Pharm Biomed Anal. Quality assessment of Cinnamomi Ramulus by the simultaneous analysis of multiple active components using high-performance thin-layer chromatography and high-performance liquid chromatography. J Sep Sci. Fingerprint analysis and multi-ingredient determination using a single reference standard for Saposhnikoviae Radix.
Anal Sci. Anal Methods. A reproducible analytical system based on the multi-component analysis of triterpene acids in Ganoderma lucidum. Simultaneous HPLC quantification of five major triterpene alcohol and sterol ferulates in rice bran oil using a single reference standard. Food Chem. Simultaneous determination of four active components in Alisma orientale Sam. J Pharmaceut Anal. Column selectivity in reversed-phase liquid chromatography: IV type-B alkyl-silica columns.
Column selectivity in reversed-phase liquid chromatography: V. Higher metal content type-A alkyl-silica columns. Column selectivity in reversed-phase liquid chromatography: VI. Columns with embedded or end-capping polar groups. Characterisation of reversed-phase liquid chromatographic columns by chromatographic tests: rational column classification by a minimal number of column test parameters. Characterisation of reversed-phase liquid-chromatographic columns by chromatographic tests: comparing column classification based on chromatographic parameters and column performance for the separation of acetylsalicylic acid and related compounds.
HPLC column classification.
Method Development and Validation Parameters of HPLC- A Mini Review | Open Access Journals
Pharm Forum. Choosing an equivalent replacement column for a reversed-phase liquid chromatographic assay procedure. The hydrophobic-subtraction model of reversed-phase column selectivity. Facilitated column selection in pharmaceutical analyses using a simple column classification system.
Column selection for pharmaceutical analyses based on a column classification using four test parameters. Evaluation of two approaches to characterise liquid chromatographic columns using pharmaceutical separations. A new method to improve the reproducibility of retention time on reversed phase C 18 columns in different laboratories. Chin J Anal Chem. Two reference substances for determination of multiple components 1 : linear calibration using two reference substances for identification of chromatographic peaks.
The acronyms referred by you relate to detectors for Gas Chromatography. FPD- Flame photometric detector. The query raised by you is mainly a method development issue. You will be fortunate if you come across a standard operating procedure based on a validated method using a technique with the right choice of technique combined with a suitable column and detector. In absence of any such method you can develop your own in-house method depending on availability of resources. However, for proficiency level testing in different laboratories there has to be the same method of analysis using identical set of conditions.
I am sending it to some friends ans additionally sharing in delicious. And certainly, thank you in your effort! But tell me can i use this comparison in my examination , the question which asked in past paper is. Please tell me? Thank you so much for this explaination. So, can you explain why we use hplc instead of gcms to analyse antibiotic? Both phases will not be same. You have to refer to supplier catalogs of columns or also refer to applications in research journals to arrive at your choice of suitable columns.
Thank you so much for this clear difference. Everyone who read this, must encourage you for this amazing work. Nice and Informative post, thank you such a nice sharing we can hope find more from you like this topic, thanks again. Over scientists read our free weekly newsletter. Don't be left out! Deepak Bhanot. Gas Chromatograph. High Performance Liquid Chromatograph. About Dr. Deepak Bhanot Dr Deepak Bhanot is a seasoned professional having nearly 30 years expertise beginning from sales and product support of analytical instruments.
Comments Girish Kulkarni says: April 2, at pm. Dear Dr. Thanks for all your articles……… Regards Girish Kulkarni. Deepak Bhanot says: April 3, at am. Hello Deepak Sir.. Best Regards K. Deepak Bhanot says: August 2, at pm.