19 High performance thin layer chromatography (HPTLC)
Dr. Varinder Kaur
Objectives: To study the basics of high performance thin layer chromatography and know the following questions.
1. What is HPTLC?
2. Why is it needed?
3. How it works?
4. What is the difference between TLC and HPTLC?
1. Description
The chromatographic techniques showed continuous improvement beginning from use of starch as a binder, silicic acid as sorbent, and use of silica as stationary phase in TLC. The TLC exhibited wide applicability in the separation science and gained quick response throughout the world because of its important features like ability to modulate the layer thickness, layer uniformity, binding sites, and controlling pore size, volume, specific surface area and particle size of sorbent. It was commercialized in the form of pre-coated TLC plates and considered as a fast and cost-effective technique for separation purposes. After the establishment of silica coated TLC as an efficient tool for quantitative techniques, effect of small sized silica coatings on Rf values and plate height were observed. The plates with small sized silica (termed as nano-plates) were found advantageous and were called as HPTLC plates. This led to the introduction of high performance thin layer chromatography (HPTLC).
It is a type of planar (flat bed) chromatography and is sophisticated version of TLC with the enhancements like increased resolution of components, use of higher quality plates and small sized stationary phase. The modern HPTLC technique involves automated sample application and densitometric scanning. It is highly sensitive and is suitable for both the qualitative and quantitative analysis. It is better over other chromatographic techniques as it provides fingerprints for visualization and ability to store data in the form electronic library.
2. Principle
The principle of HPTLC is same as TLC (i.e. separation by adsorption). In this technique, mobile phase is driven by capillary action and carries various components. Depending on their affinity towards adsorbent, the components exhibit adsorption on the stationary phase. The components with larger efficiency for stationary phase interact with the surface and move slowly in the mobile phase whereas components with lesser affinity for stationary phase move fast. This variation in the movement of components results in the separation of components on the chromatographic plate.
3. Advancement of HPTLC over TLC
Although HPTLC works on the same principle as TLC, however, it offers advantages because it is robust, simple, rapid, efficient in quantitative analysis, gives better resolution and limit of detection.
Some of the important features of TLC and HPTLC are compared below.
4. Key features of HPTLC
Some of the important features of HPTLC are given below.
1. It produces complex information about the entire sample in the form of visible chromatograms at a glance.
2. Simultaneously, sample and standard can be analyzed for better precision and accuracy.
3. Various samples can be analyzed and compared simultaneously with the help of images.
3. It gives data in the form of visible chromatograms as well as peak data.
4. The data can be evaluated either by the image based software Videoscan or by scanning densitometry with TLC Scanner, measuring the absorption and/or fluorescence of the substances on the plate.
5. The technique is cost-effective and has low cost for maintenance.
6. The sample preparation is simple and different types of samples can be analyzed by the technique.
7. Prior treatment of solvents i.e. filtration and degassing is not required.
8. The use of harmful solvents is less as compared to other chromatographic techniques.
9. The chances of contamination are less because it utilizes fresh stationary and mobile phases for each analysis.
5. Working of HPTLC
The various steps of HPTLC are discussed below.
Selection of HPTLC chromatographic layer and mobile phase
In HPTLC, plates coated with small sized particles and narrow size distribution is used. Thus, the surface area of plates is smooth. The size of the plates is comparatively small as compared to TLC i.e. (10 10-20 cm) and the development distance is 6 cm. The selection of HPTLC stationary phase is based upon the type of analyte. The most common HPTLC stationary phases are listed below.
A care must be taken while handling the plates to avoid contamination. The plates should be observed under UV light to know any damage or presence of impurities on the adsorbent. The HPTLC plates should be prewashed to remove the impurities (if any) adsorbed on their surface. However, pre-washing is required to study the reproducibility of results and for quantification purposes. The washing may be done by ascending, dipping or continuous method. The most common solvents used for washing purposes are methanol, chloroform or their mixtures. The plates kept in the open must be activated by placing in an oven at 110-130 C for 30 min.
The selection of mobile phase is made on the basis of type of adsorbent, and physical and chemical properties of analyte.
5.1 Sample preparation
The samples may be prepared methanol, chloroform:methanol (1:1 v/v), ethyl acetate: methanol (1:1 v/v), chloroform:methanol:ammonia (90:10:1 v/v), methylene chloride:methanol (1:1 v/v) to avoid interference from impurities and water vapours.
5.2 Application of sample
The sample is applied with the help of some applicators such as 1) capillary tubes 2) micro-bulb pipettes, 3) micro-syringes, and 4) automated sample applicators. Using these applicators, sample can be applied in the form of spot or band. The concentration range is 0.1-1µg /µL because above this range, separation becomes poor.
In case of applying a sample as a spot, a fixed volume pipette having capillary action is used. It is filled with the sample and touched on the surface of plate, thereby delivering the sample to the stationary layer. If variable volumes are needed, then, a syringe with micrometer control can be used. Proper care must be taken during the application of sample i.e. the spots should be precise in position and layer should not undergo any damage during application. In sample applicators, Camag Nanomat is a mechanized spotting device with fixed volume glass capillaries. These are lowered onto the layer with reproducible contact pressure (Figure 1), which controls the position of the spot. Similarly, Desaga PS 01 Sample Applicator uses microlitre syringes for the application of sample. Another method i.e. sample application as narrow bands provides the highest resolution in the separation. In this case, sample is filled in a syringe and vacated with the help of a motor. Both the syringe and plate move linearly to produce a band. Another applicator i.e. Linomat (Figure 2) allows sample application in narrow bands of variable length.
Apart from this, automatic samplers are computer controlled and can apply samples automatically. The samples are picked from the vials and transferred to plate with the help of steel capillary in the form of spots or bands. These devices offer choice to select the sample volume, dispensing speed and application pattern.
5.3 Chromatograph development
The chromatogram development is the most important step in the HPTLC procedure. The HPTLC chamber is pre-conditioned with solvent to get uniform vapors of the solvent in the chamber. The chromatograms can be developed in four ways 1) Vertical method, 2) Vario-method, 3) horizontal method and 4) automatic multiple development. The HPTLC plates are generally developed in twin-trough chambers, or horizontal-development chambers. The saturated twin-trough chambers fitted with filter paper offers the best reproducibility and avoids solvent vapor preloading and humidity.
In the cortical method, the lower edge of the plate is immersed in the developing chamber having solvent at the bottom. The solvent ascends in the plate and layer interacts with the vapours in the tank. This method does not provide reproducibility as the development of chromatograms varies with the dimensions of the plate. In the horizontal development, chromatogram is developed by applying the sample parallel to both opposing edges of the plate. The chromatogram is developed from both the sides towards the centre of the plate. Therefore, the number of samples can be doubled. In this method, the volume of solvent required for the development is very less, therefore, it is economical. In automatic development, the development of chromatogram can be controlled by an instrument. In offers the advantage to select various parameters like preconditioning, tank of sandwich configuration, solvent migration distance, etc prior to the development of chromatogram. In this case, software is used to decide the composition of developing solvent and developing distance. In addition, the volumes are measured with syringes and migration distance can be measured by sensors.
5.4 Detection of spots (Scanning) and documentation
The developed plates can be detected by using UV cabinet or chamber which provides a non- destructive analysis. Alternatively, the spots are analyzed at 254 nm or 366 nm if the compounds are fluorescent. Moreover, fluorescent stationary phases may be used to if the compounds exhibit quenching properties. These days, design of UV cabinets is improved, which allows fixing of digital camera for recording images of the plate. Further, the components may be quantified on the same plates.
5.5 Densitometer measurements
In densitometery, separation tracks are evaluated with the help of a light beam in the form of a slit with adjustable dimensions. The reflected light is measured by the photosensor and the difference between optical response of blank and the sample zone is correlated with various sample zones. Nowadays, a planar chromatogram is evaluated by video technology.
6. Coupling of HPTLC with other techniques
The coupling of planar chromatography with various chromatographic or non-chromatographic methods has been reported, which includes HPLC-TLC, TLC-FTIR, TLC-Raman, TLC-SERS and TLC-MS.
6.1 Coupling of HPTLC with HPLC
The HPTLC can be coupled with HPLC by online coupling. The combination of HPLC with multiple development system results in peak capacity of around 500. It consists of a sample spray-on device connected to the outlet of the column. This combination is highly useful..
6.2 Coupling of HPTLC with FTIR
This combination is very useful for the identification of complex mixtures and their constituents. The substances insensitive to UV detection as well as difficult to derivatize can be easily evaluated by this method. But using in situ FTIR, a major drawback is strong absorption of silica gel between 3700-3100 and 1650-800 cm-1.
6.3 Coupling of HPTLC with Raman spectroscopy
Surface enhanced Raman scattering (SERS) is more suitable for the identification of substances on a TLC plate as compared to regular Raman Spectroscopy due to its detection in picogram range if the layer is treated with a colloidal silver solution, as compared to 0.5-5 µg per fraction in Raman spectra.
6.4 Coupling of HPTLC with Mass Spectrometry
HPTLC coupled with mass spectrometry is mainly studied by using electrospray ionization (ESI) techniques. Desorption of the molecules from the layer and then introduction into the ion source of the mass spectrometer is required for the in situ identification of compounds. The laser ablation or particle beam sputtering techniques can be used for desorption. The detection is in the pg/zone-range. Recently, planar chromatography coupled to mass spectrometry via atmospheric pressure glow discharge (APGD). The HPTLC/MS coupling offers exclusive advantages because of the local fixation of separated substance zones on the plate which means that the MS equipment and recording can be employed in a highly targeted way with reduced costs and storage of data. The main limitation is non-availability of MS instrument that takes 10 cm x 10 cm or larger plates.
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