High-performance thin-layer chromatography

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Picture of HPTLC plate to identify different substances within a sample. Hawthorn tincture HPTLC Identification.jpg
Picture of HPTLC plate to identify different substances within a sample.

High-performance thin-layer chromatography (HPTLC) serves as an extension of thin-layer chromatography (TLC), offering robustness, simplicity, speed, and efficiency in the quantitative analysis of compounds. [1] This TLC-based analytical technique enhances compound resolution for quantitative analysis. Some of these improvements involve employing higher-quality TLC plates with finer particle sizes in the stationary phase, leading to improved resolution. [2] Additionally, the separation can be further refined through repeated plate development using a multiple development device. As a result, HPTLC provides superior resolution and lower Limit of Detection (LODs). [3]

Contents

Instrumentation

Advantages of HPTLC: [1]

Mode

HPTLC comprises three modes: linear mode, circular mode, and anticircular mode. Among these modes, the anticircular mode stands out as the fastest in theory and practice within the realm of HPTLC. This mode achieves separation by allowing the mobile phase to enter the plate layer precisely along an outer circular path, after which it flows toward the center at a nearly constant speed. This approach maximizes sample capacity while minimizing time, layer, and mobile phase consumption, making it the most cost-effective HPTLC technique. The narrow spot-path unique to anticircular HPTLC facilitates automated quantification. When compared to the linear and circular modes, the anticircular mode demonstrates superior separation and significantly heightened sensitivity, especially at higher Rf-values. [2]

Methodology

To begin HPTLC, a stationary phase has to be determined to separate different compounds within a mixture. Around 90% of all pharmaceutical separations are performed on normal phase silica gel; however, other stationary phases such as alumina can be used for samples with dissociating compounds and cellulose for ionic compounds. [4] The reverse-phase HPTLC method (similar methodology to reverse-phase TLC) is used for compounds with high polarity. After the selection of the stationary phase, plates are generally washed with methanol and dried in an oven to remove excess solvent. [5]

Selection for the mobile phase is one of the most important processes of HPTLC and follows a 'trial and error' pathway. However, the 'PRISMA' system stands as a guideline for finding the optimal mobile phase. [1] The mobile phase is dependent on the absorptivity of the stationary phase and the composition of the compound of interest. [5] The compound is first tested with solutions such as diethyl ether, ethanol, dichloromethane, chloroform for normal phase HPTLC, or solutions such as methanol, acetonitrile, and tetrahydrofuran for reverse phase HPTLC. The retardation factors (Rf) of the compounds with the selected solvent are then analyzed and the solvent that gives the largest Rf is chosen to be the mobile phase for the compound. Then, the mobile solvent strength is tested against hexane (for normal HPTLC) and water (for reverse-phase HPTLC) to determine the need for adjustment. [5] [6]

CAMAG HPTLC machine Typical HPTLC Instruments.jpg
CAMAG HPTLC machine

Notable HPTLC devices such as the Linomat 5 and the Automatic TLC Sampler 4 (ATS 4) by CAMAG function very similarly by having the automated 'spray-on' sample application technique. [4] [5] This automated 'spray-on' technique is useful to overcome the uncertainty in droplet size and position when the sample is applied to the TLC plate by hand. Additionally, automation provides high resolution and narrow bands since the solvent evaporates immediately as the sample makes contact with the plate. [4] One approach to automation has been the use of piezoelectric devices and inkjet printers for applying the sample. [7] Alternatively, the Nanomat 4 and ATS 4 by CAMAG are manually operated where the sample is applied via spot application using a capillary pipette. [4] [5]

Upon chromatographic detection, HPTLC plates are usually developed in saturated twin-trough chambers with filter paper for optimal outcomes. [5] [6] However, flat-bottom chambers and horizontal-development chambers are also used for specific compounds. A general mechanism for the HPTLC device goes as follows. [5] A fitted filter paper is placed in the rear trough of the chamber and the mobile phase is poured through the rear trough to ensure complete solvent absorption of the filter paper. The chamber is then tilted to ~45° so both troughs are equal in solvent volume and left alone to equilibrate for ~20 mins. [5] Finally, the HPTLC plate is placed in the chamber to develop. Between each sample reading, the mobile phase and filter paper are changed to ensure the best outcomes.

The spot capacity (analogous to peak capacity in HPLC) can be increased by developing the plate with two different solvents, using two-dimensional chromatography. [8] The procedure begins with development of a sample loaded plate with first solvent. After removing it, the plate is rotated 90° and developed with a second solvent.

Applications

HPTLC finds extensive application in various fields, including pharmaceutical industries, clinical chemistry, forensic chemistry, biochemistry, cosmetology, food and drug analysis, environmental analysis, and more, owing to its numerous advantages. It distinguishes itself by being the only chromatographic method capable of presenting results as images and offers simplicity, cost-effectiveness, parallel analysis of samples, high sample capacity, rapid results, and the option for multiple detection methods.

Le Roux's research team assessed HPTLC for determining salbutamol serum levels in clinical trials and concluded that it is a suitable method for analyzing serum samples. [3]

HPTLC has also been used successfully in the separation of various lipid subclasses, with reproducible and promising results obtained for 20 different lipid subclasses. Numerous reports related to clinical medicine studies have been published in various journals. As a result, HPTLC is now strongly recommended for drug analysis in serum and other tissues. [7]

Related Research Articles

In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent called the mobile phase, which carries it through a system on which a material called the stationary phase is fixed. Because the different constituents of the mixture tend to have different affinities for the stationary phase and are retained for different lengths of time depending on their interactions with its surface sites, the constituents travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.

<span class="mw-page-title-main">High-performance liquid chromatography</span> Technique in analytical chemistry

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 specific components in mixtures. The mixtures can originate from food, chemicals, pharmaceuticals, biological, environmental and agriculture, etc, which have been dissolved into liquid solutions.

<span class="mw-page-title-main">Gas chromatography</span> Type of chromatography

Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.

<span class="mw-page-title-main">Paper chromatography</span> Separation of coloured chemicals on paper

Paper chromatography is an analytical method used to separate coloured chemicals or substances. It is now primarily used as a teaching tool, having been replaced in the laboratory by other chromatography methods such as thin-layer chromatography (TLC).

<span class="mw-page-title-main">Column chromatography</span> Method to isolate a compound in a mixture

Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.

<span class="mw-page-title-main">Ion chromatography</span> Separates ions and polar molecules

Ion chromatography is a form of chromatography that separates ions and ionizable polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including small inorganic anions, large proteins, small nucleotides, and amino acids. However, ion chromatography must be done in conditions that are one pH unit away from the isoelectric point of a protein.

<span class="mw-page-title-main">Thin-layer chromatography</span> Technique used to separate non-volatile mixtures

Thin-layer chromatography (TLC) is a chromatography technique that separates components in non-volatile mixtures.

<span class="mw-page-title-main">Liquid chromatography–mass spectrometry</span> Analytical chemistry technique

Liquid chromatography–mass spectrometry (LC–MS) is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography with the mass analysis capabilities of mass spectrometry (MS). Coupled chromatography – MS systems are popular in chemical analysis because the individual capabilities of each technique are enhanced synergistically. While liquid chromatography separates mixtures with multiple components, mass spectrometry provides spectral information that may help to identify each separated component. MS is not only sensitive, but provides selective detection, relieving the need for complete chromatographic separation. LC–MS is also appropriate for metabolomics because of its good coverage of a wide range of chemicals. This tandem technique can be used to analyze biochemical, organic, and inorganic compounds commonly found in complex samples of environmental and biological origin. Therefore, LC–MS may be applied in a wide range of sectors including biotechnology, environment monitoring, food processing, and pharmaceutical, agrochemical, and cosmetic industries. Since the early 2000s, LC–MS has also begun to be used in clinical applications.

Chiral column chromatography is a variant of column chromatography that is employed for the separation of chiral compounds, i.e. enantiomers, in mixtures such as racemates or related compounds. The chiral stationary phase (CSP) is made of a support, usually silica based, on which a chiral reagent or a macromolecule with numerous chiral centers is bonded or immobilized.

Reversed-phase liquid chromatography (RP-LC) is a mode of liquid chromatography in which non-polar stationary phase and polar mobile phases are used for the separation of organic compounds. The vast majority of separations and analyses using high-performance liquid chromatography (HPLC) in recent years are done using the reversed phase mode. In the reversed phase mode, the sample components are retained in the system the more hydrophobic they are.

<span class="mw-page-title-main">Hydrophilic interaction chromatography</span> Type of chromatography

Hydrophilic interaction chromatography is a variant of normal phase liquid chromatography that partly overlaps with other chromatographic applications such as ion chromatography and reversed phase liquid chromatography. HILIC uses hydrophilic stationary phases with reversed-phase type eluents. The name was suggested by Andrew Alpert in his 1990 paper on the subject. He described the chromatographic mechanism for it as liquid-liquid partition chromatography where analytes elute in order of increasing polarity, a conclusion supported by a review and re-evaluation of published data.

Supercritical fluid chromatography (SFC) is a form of normal phase chromatography that uses a supercritical fluid such as carbon dioxide as the mobile phase. It is used for the analysis and purification of low to moderate molecular weight, thermally labile molecules and can also be used for the separation of chiral compounds. Principles are similar to those of high performance liquid chromatography (HPLC); however, SFC typically utilizes carbon dioxide as the mobile phase. Therefore, the entire chromatographic flow path must be pressurized. Because the supercritical phase represents a state whereby bulk liquid and gas properties converge, supercritical fluid chromatography is sometimes called convergence chromatography. The idea of liquid and gas properties convergence was first envisioned by Giddings.

Micellar liquid chromatography (MLC) is a form of reversed phase liquid chromatography that uses an aqueous micellar solutions as the mobile phase.

Aqueous normal-phase chromatography (ANP) is a chromatographic technique that involves the mobile phase compositions and polarities between reversed-phase chromatography (RP) and normal-phase chromatography (NP), while the stationary phases are polar.

In chromatography, the retardation factor (R) is the fraction of an analyte in the mobile phase of a chromatographic system. In planar chromatography in particular, the retardation factor RF is defined as the ratio of the distance traveled by the center of a spot to the distance traveled by the solvent front. Ideally, the values for RF are equivalent to the R values used in column chromatography.

<span class="mw-page-title-main">Two-dimensional chromatography</span>

Two-dimensional chromatography is a type of chromatographic technique in which the injected sample is separated by passing through two different separation stages. Two different chromatographic columns are connected in sequence, and the effluent from the first system is transferred onto the second column. Typically the second column has a different separation mechanism, so that bands that are poorly resolved from the first column may be completely separated in the second column. Alternately, the two columns might run at different temperatures. During the second stage of separation the rate at which the separation occurs must be faster than the first stage, since there is still only a single detector. The plane surface is amenable to sequential development in two directions using two different solvents.

Partition chromatography theory and practice was introduced through the work and publications of Archer Martin and Richard Laurence Millington Synge during the 1940s. They would later receive the 1952 Nobel Prize in Chemistry "for their invention of partition chromatography".

<span class="mw-page-title-main">Countercurrent chromatography</span>

Countercurrent chromatography is a form of liquid–liquid chromatography that uses a liquid stationary phase that is held in place by inertia of the molecules composing the stationary phase accelerating toward the center of a centrifuge due to centripetal force and is used to separate, identify, and quantify the chemical components of a mixture. In its broadest sense, countercurrent chromatography encompasses a collection of related liquid chromatography techniques that employ two immiscible liquid phases without a solid support. The two liquid phases come in contact with each other as at least one phase is pumped through a column, a hollow tube or a series of chambers connected with channels, which contains both phases. The resulting dynamic mixing and settling action allows the components to be separated by their respective solubilities in the two phases. A wide variety of two-phase solvent systems consisting of at least two immiscible liquids may be employed to provide the proper selectivity for the desired separation.

Chiral analysis refers to the quantification of component enantiomers of racemic drug substances or pharmaceutical compounds. Other synonyms commonly used include enantiomer analysis, enantiomeric analysis, and enantioselective analysis. Chiral analysis includes all analytical procedures focused on the characterization of the properties of chiral drugs. Chiral analysis is usually performed with chiral separation methods where the enantiomers are separated on an analytical scale and simultaneously assayed for each enantiomer.

<span class="mw-page-title-main">Chiral thin-layer chromatography</span>

Chiral thin-layer chromatography is a variant of liquid chromatography that is employed for the separation of enantiomers. It is necessary to use either

References

  1. 1 2 3 Morlock, Gertrud E. (2 October 2021). "High-performance thin-layer chromatography combined with effect-directed assays and high-resolution mass spectrometry as an emerging hyphenated technology: A tutorial review". Analytica Chimica Acta. 1180: 338644. doi: 10.1016/j.aca.2021.338644 . ISSN   0003-2670. PMID   34538319. S2CID   236348479.
  2. 1 2 Kaiser, R. E. (September 1978). "Anticircular high performance thin‐layer chromatography". Journal of High Resolution Chromatography. 1 (3): 164–168. doi:10.1002/jhrc.1240010309. ISSN   0935-6304.
  3. 1 2 Le Roux, A.M.; Wium, C.A.; Joubert, J.R.; Van Jaarsveld, P.P. (October 1992). "Evaluation of a high-performance thin-layer chromatographic technique for the determination of salbutamol serum levels in clinical trials". Journal of Chromatography B: Biomedical Sciences and Applications. 581 (2): 306–309. doi:10.1016/0378-4347(92)80288-2. ISSN   0378-4347. PMID   1452625.
  4. 1 2 3 4 Shewiyo, D. H.; Kaale, E.; Risha, P. G.; Dejaegher, B.; Smeyers-Verbeke, J.; Heyden, Y. Vander (1 July 2012). "HPTLC methods to assay active ingredients in pharmaceutical formulations: A review of the method development and validation steps" . Journal of Pharmaceutical and Biomedical Analysis. 66: 11–23. doi:10.1016/j.jpba.2012.03.034. ISSN   0731-7085. PMID   22494517.
  5. 1 2 3 4 5 6 7 8 Patel, Rashmin B.; Patel, Mrunali R.; Batel, Bharat G. (2011), Srivastava, ManMohan (ed.), "Experimental Aspects and Implementation of HPTLC", High-Performance Thin-Layer Chromatography (HPTLC), Berlin, Heidelberg: Springer, pp. 41–54, doi:10.1007/978-3-642-14025-9_3, ISBN   978-3-642-14025-9 , retrieved 6 November 2023
  6. 1 2 Sherma, Joseph (1 May 2010). "Review of HPTLC in Drug Analysis: 1996-2009". Journal of AOAC International. 93 (3): 754–764. doi: 10.1093/jaoac/93.3.754 . ISSN   1060-3271. PMID   20629372 . Retrieved 6 November 2023.
  7. 1 2 Bernardi, Tatiana; Tamburini, Elena (October 2009). "An HPTLC-AMD method for understanding the metabolic behavior of microorganisms in the presence of mixed carbon sources. The case ofBifidobacterium adolescentisMB 239". Journal of Planar Chromatography – Modern TLC. 22 (5): 321–325. doi:10.1556/jpc.22.2009.5.2. ISSN   0933-4173. S2CID   86407898.
  8. ↑ Nurok, David (1 March 1989). "Strategies for optimizing the mobile phase in planar chromatography". Chemical Reviews. 89 (2): 363–375. doi:10.1021/cr00092a007. ISSN   0009-2665.
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