Technology Forum: HPLC/TLC

This month, Chromatography Online's Technology Forum looks at the topic of HPLC/TLC and the trends and issues surrounding it. Joining us for this discussion are Eike Reich from Camag, Gerda Morlock, from University of Hohenheim, and Colin poole, from Wayne University.

What has been one of the most important advances in past decade of thin-layer chromatography documentation? How has this advancement changed thin-layer chromatography? What is the significance of this advancement? Are there any other significant documentation achievements in thin-layer chromatography that you'd like to mention?

Reich: In the last decade documentation of TLC chromatograms with digital cameras became possible. Most recently I would consider the availability of an affordable and dedicated documentation system, which features a high performance 12 bit industrial CCD camera* with standardized capture settings, as the most important advancement. This system can be qualified for use in a regulated environment such as cGMP. In this qualification a calibration can be performed and any non-homogeneity of illumination is corrected so that true (in the sense of realistic, lifelike) images of the HPTLC plate can be obtained. Such images are highly reproducible, secure against adulteration, and independent of the operator. They can be the basis for comparison of TLC results from day to day or lab to lab. Images can now be used reliably as reference for example in quality control of raw materials, stability studies over long time periods, and screening of large numbers of samples. Modern software allows comparison of many images as well as of individual chromatogram tracks coming from different HPTLC plates.But there is another aspect. True, reproducible, high quality images open the door to accurate and precise quantitative determinations through video** densitometry, which most conveniently complements the classic scanning densitometry. It is now possible to enhance the chromatographic results by subtracting images of the neat plate (prior to sample application) from images of the developed plate, a process which eliminates most of the background noise. Of course all this can only be fully appreciated in connection with advancements concerning the entire HPTLC process, of which documentation is only the final step. I'd like to mention new concepts of standardization of the HPTLC methodology as well as groundbreaking new approaches in validating the performance criteria of qualitative HPTLC methods. Without those we would just accurately document poor chromatography.

*as opposed to high-end professional consumer cameras used in photography

** this term is commonly used to describe the process of transferring image data into analogue chromatogram curves

Morlock: The situation for planar chromatography changed from poor reputation for unsatisfactory reproducibility to a standardized reproducible and quantitative method. Thanks to devoted analysts and companies who took care about the conditions ensuring reproducibility and reliability, the method turned out to be much more than just a simple screening tool. Investment in technology turned planar chromatography into an in all steps automated and standardized method with a high degree of flexibility. For example, it is possible nowadays - due to automated chambers with integrated humidity control - to obtain the same chromatogram all over the world over a wide time period despite differences in the humidity of the air. New documentation systems based on 12 bit cameras with a highly linear CCD chip allow excellent color reproduction and time-effective evaluation or image comparison of the reproducible high-performance photos. In the age of ultra rapid separations, especially planar chromatography is an issue: 44 parallel chromatographic runs under identical conditions allow separations in the 20-seconds stroke with a mobile phase consumption of 0.4 mL per run. After quantification the positive findings - often the minority of all the analytical determinations - can cost-effectively be confirmed by mass spectrometry automated within 2 min per run. Thus the method itself was capable all over the years; however, even today scant attention is paid to ensure proper conditions and the necessary investment essential for the reliability of the method. Changing attitudes might help "to keep analysis cheap" for a variety of issues even now the analytical challenges increase.

Poole: For documentation video densitometry has been the most important development in the last decade. Scanning densitometry remains the gold standard for quantification but video densitometry provides adequate results for many applications combined with the power of images to inform and the wide range of dedicated and general software that allow images to be shared, sorted and searched. The area of fingerprinting, especially authentication and quality control of herbal products, has benefited most from the introduction of video densitometry.

There are many current trends in thin-layer chromatography such as forced flow techniques and video densitometry. In your opinion, which trend is the most important for the future of thin-layer chromatography? How will this technique help analytical scientists? What obstacles currently block further development of this advancement? What other trends do you think will be important in the future of thin-layer chromatography? Could you list any other current/future trends in thin-layer chromatography that our readers should know?

Reich: Gradient techniques such as Automated Multiple Development and forced flow techniques using pump pressure, shear forces, or electric fields are certainly interesting developments. Also attempts to miniaturize the chromatographic system while increasing the separation efficiency as seen in UTLC and circular HPTLC on small particles should be followed closely. However I am convinced that there is still a huge and diverse field of applications to open up for "regular" High Performance Thin Layer Chromatography once analysts realize the enormous potential of a standardized approach and the consequent use of state of the art instrumentation. I have already mentioned video densitometry in connection with electronic images as a powerful tool for quantitative analysis. In addition to the documentation step and subsequent image evaluation I see the recent advances in chromatogram development as the most fundamental accomplishment. Today HPTLC plates can be automatically developed without interference of any environmental or manual factor. Three of the most critical parameters of TLC are now under complete control; the composition (saturation) of the gas phase in the chromatographic chamber, the activity of the stationary phase (as function of relative humidity), and the homogenous drying of the plate after development. It is possible to get exactly the same predictable result in different labs/parts of the world, if the same sample is analyzed with the same method. This is the basis for future official or compendial HPTLC methods of analysis. This is the basis for regaining trust in a method that in its classical "quick, cheap, and simple" version has lost a lot of ground to the "almighty" HPLC. HPTLC today is very powerful and it is an orthogonal approach to HPLC. Adsorption vs. partition, off-line vs. on-line, and open system vs. black box are only a few keywords that support the idea that HPTLC should be seen as the perfect compliment to HPLC.

What are the obstacles? Primarily I see inertia. TLC has always been quick, simple, cheap. It was an official method when there was no HPLC. Although HPTLC plates were introduced in the late seventies, it took until the turn of the century before a broader recognition of the merits of instrumental HPTLC as an analytical concept was seen. Many analysts have never been acquainted with it and have not yet experienced the new possibilities. And yes, initial costs of HPTLC are significant, but so are those of LC-MS, NIR, NMR and other instrumental methods. Running costs and cost per sample on the other hand are very low. Good tools have their price but if the result turns out as expect, it is all relative.

For the near future I see the commercialization of already existing interfaces for hyphenation of HPTLC and mass spectrometry. I expect an increase in the use of biological tests on the developed HPTLC plate as way of screening separated compound for biological activity. Last but not least I think we will soon see miniaturized fully automatic HPTLC systems for specialized applications.

Morlock: Besides miniaturization, speed and increasing the separation power, especially the coupling of planar chromatography with mass spectrometry is an important feature. In contrast to column techniques, the automated, off-line planar chromatographic method allows evaluation first. Then mass spectrometry can be utilized just for "hot" zones allowing an immense cost-reduction. Extremely helpful for advanced analysts is the flexibility of the method. It is impressive time after time, particular for solving difficult problems in a simple way. For example complex matrix can be left at the starting zone on the plate, allowing reduced sample preparation, but generally not on a column. Selective derivatizations solve analytical tasks which could otherwise only be managed by HPLC/MS-MS. However, first analysts must recognize the benefits of the high-performance employment of the method and secondly they must dare the impossible to use a method not in the mainstream. Few analysts had overcome these obstacles so far.

The myriad of chemicals and progressive sensitivity of the analytical methods, which reveal more and more secrets of the past, challenge analysts of today. However, important questions, related to toxicity or bio-activity, are rarely touched. Degradation products or metabolites can be much more toxic, but are generally of minor concern in target analysis because they are unknown or no reference standards are available. A new analytical concept of bio-activity based detection might be reasonable where planar chromatography is an essential tool. The planar storage of separated runs enables bio-activity-based detection of single substances which are generating a distinct effect. This concept is at the very beginning, but surely accounts for food safety concerns or pharmaceutical issues in the future.

Poole: High-performance or instrumental TLC has never developed in the way that many of us expected. In its most advanced form high-performance TLC requires a significant investment in instrumentation that many laboratories never made. One reason is its lower performance compared with column chromatography. Forced flow development and electrochromatography where the approaches supposed to change this situation. Quite good equipment is now available for forced flow development but electrochromatography seems to have hit the buffers (somewhat like electrochromatography in column chromatography). The reputation of forced flow development was injured by the poor quality of early instrumentation that was barely usable for routine applications. Too much hype did not help either, and it became an invisible tool before it reached a useful level of maturity.

The most recent development in TLC that excites me is the use of biological detection. A kit is now available for the postchromatographic application of fluorescent bacteria to layers for the detection of toxic zones. There are a number or reports, largely from Europe, on the screening of wastewater samples for toxic compounds employing two-dimensional TLC followed by detection of bacterial fluorescence inhibition to qualify the nature of any chemical hazards. This approach is quite flexible and the number of potential indicator reagents/species that could be used for different target compounds is very large. Biological response detection affords a new dimension to analysis by facilitating the analysis of samples based on risk rather than the current target compound approach.

In regard to this type of approach recent advances in the direct coupling TLC to mass spectrometry are timely. The evolution of this field has introduced a number of partially manual techniques that are difficult to automate. Recently two research groups have described different approaches for the automated scanning of a TLC plate with the realization of mass spectra from zones of interest. There is a reasonable chance that these interfaces will become commercially available since they are easily adapted to standard mass spectrometer hardware. Quantitative analysis at low levels of selected ions has also been indicated. The identification of unknown zones in TLC has been something of a problem in the past. Reasonable solutions to do this quickly and as easily as for column chromatography seem to be just around the corner.