Session 7

 1 Irish Separation Science Cluster (ISSC), Department of Chemistry and the Analytical &Biological Chemistry Research Facility (ABCRF), University College Cork, Western Road, Cork, Ireland.

2 Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec, Canada H4P2R2, This email address is being protected from spambots. You need JavaScript enabled to view it.

Increasingly nanoscale particles, pores and surfaces, are being tailor-made to provide high performance separation and detection. New superficially porous (core shell) silica particles for liquid chromatography are a good example in separation science, where nanoscale control over shell thickness enhances column efficiency. Nanoparticle incorporation in the separation and/or sensing roles, can also be an attractive means of enhancing selectivity and detectability.

In this work, sensitive and selective detection of disease indicating biomarkers is demonstrated using capillary electrophoresis with embedded gold and silica nanoparticles and amperometric detection at a boron doped diamond (BDD) electrode. The BDD electrode has emerged to play an important role in improving sensitivity in electrochemical detection. Luong et al.1 reviewed the application of the BDD electrode for sensitive detection in bioanalysis. Chen et al.2 reviewed the application of the BDD electrode for electrochemical detection in capillary electrophoresis.

By coating the fused silica capillary with gold nanoparticles (AuNPs) embedded in a cationic polymer poly (diallyldimethylammonium) chloride (PDDA), efficient separation and sensitive detection was achieved for analysis of 3-indoxyl sulphate (IXS), vanillylmandelic acid (VMA), homovanillic acid (HVA) and tryptophan (TRP) in urine3. The application of this separation and sensing approach is further applied in the field of bacterial cell-to-cell communication in the analysis of cell signalling molecules in the Pseudomonas Quinolone Signal (PQS) quorum sensing system4. P. aeruginosa is best known as an antibiotic resistant human pathogen associated with hospital-acquired infections and is the primary cause of morbidity and mortality in cystic fibrosis sufferers.

References:

1. J.H.T. Luong, K.B. Male, J.D. Glennon, Analyst, 2009, 134, 1965.
2. G. Chen, Talanta, 2007, 74, 326.
3. L. Zhou, J.D. Glennon, J.H.T. Luong, Anal Chem, 2010, 82, 6895.
4. L. Zhou, J.D. Glennon, F.J. Reen, F. O’Gara, C.M. McSweeney, G.P. McGlacken, Chem Comm, 2011, 47, 10347.

 1 Department of Chemistry, Laboratory of Analytical Chemistry, P.O. Box 55, 00014 University of Helsinki, Finland

2 Department of Engineering and Chemical Sciences, Karlstad University, 651 88 Karlstad, Sweden

3 Wihuri Research Institute, Haartmaninkatu 8, FIN-00290 Helsinki, Finland

4 National Institute for Health and Welfare, Public Health Genomics Unit, Biomedicum, Haartmaninkatu 8, FI-00251, Helsinki, Finland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Although microsystems and miniaturized instrumental techniques, which can be considered key technologies for future progress in biochemistry, biotechnology, and medicine, have inspired many scientists toward further methodological developments, there continues to be a great need for analytical systems that enable dynamic and real-time monitoring of biological processes. Among many instrumental techniques and tools employed in exploring the role of biomolecular interactions in physiological and pathological phenomena, biosensors, capillary electromigration techniques, nano liquid chromatography and microscale thermophoresis enable small sample size, low reagent consumption, and label-free analysis, all of great advantage relative to more traditional approaches such as ELISA and affinity chromatography. The strength of the interactions is determined as affinity constants, partition coefficients, retention factors, and reduced mobilities.

In this talk versatility of capillary electromigration techniques, including adsorption energy calculations, that allow the differentiation of even small differences in the binding processes, will be introduced. Furthermore the exploitation of molecularly imprinted polymers in the isolation of human biomolecules will be demonstrated. Other techniques and approaches, such as microscale thermophoresis, quartz crystalline microbalance and molecular dynamics simulation calculations provide supportive and complementary insight into the interaction mechanisms.

[1] K. Lipponen, P. W. Stege, G. Cilpa, J. Samuelsson, T. Fornstedt, M.-L. Riekkola, Anal. Chem. 83 (2011) 6040.

[2] K. Lipponen, Y. Liu, P. Wanda Stege, K. Öörni, P. T. Kovanen, M.-L. Riekkola, Anal. Biochem.424 (2012) 71.

[3] K. Lipponen, S. Tähkä, M. Kostiainen, M.-L. Riekkola, Electrophoresis 35 (2013) 1106.

[4] K. Lipponen, S. Tähkä, J. Samuelsson, M. Jauhiainen, J. Metso, G. Cilpa-Karhu, T. Fornstedt, M. Kostiainen, M.-L. Riekkola, Anal. Bioanal. Chem. 406 (2014) 4137.

[5] J. Witos, J. Samuelsson, G. Cilpa-Karhu, J. Metso, M. Jauhiainen, M.-L. Riekkola,” Analyst, 2015, DOI: 10.1039/C5AN00210A.

 M. Curie-Skłodowska University, M. Curie-Skłodowska Sq.3, 20-031 Lublin, Poland, This email address is being protected from spambots. You need JavaScript enabled to view it.

Plants are unlimited source of biologically active substances used in the treatment of many human and animal diseases. They are also area of interest in searching new drugs that can be used to treat cardiovascular problems, diabetes, Alzheimer, cancer and other civilization illnesses. Because of increasing bacterial resistance against commonly used antibiotics there is also the urgent need for new antibacterials.

Chromatography is the most popular tool in the analysis of plant constituents. While high-performance liquid chromatography (HPLC) gives the biggest separation and detection possibilities, it does not provide information on biological properties of analytes. This problem can be solved by thin-layer chromatography –direct bioautography (TLC-DB) which can be used for searching biologically active substances in very complex matrices, such plants [1]. The principle of TLC-DB is that separation, biological detection (e.g. antimicrobial) and visualization are performed directly on a TLC layer [2]. In more detail, a developed TLC plate is dipped in a suspension of microorganisms growing in a nutrient broth and incubated in a humid atmosphere. The microorganisms grow directly on the surface of a TLC plate excluding spots of antimicrobials. Visualization is usually carried out by spraying a plate with tetrazolium salt, such as MTT, which is converted by living bacteria into the purple formazan. Creamy spots appearing against a colored background, so-called inhibition zones, point to the presence of antimicrobial agents. TLC-DB was used as a bio-guiding method to point substances with antibacterial activity in Matricaria recutita L., Achillea millefolium L., Salvia officinalis L., Thymus vulgaris L., Chelidonium majus L. and Hypericum perforatum L. extracts. The targeted substances, found by TLC-DB in analytical scale, were isolated in bigger amounts using TLC semi-preparative fractionation and then subjected to LC-MS analysis for structural evaluation.

[1] Marston, A. J.Chromatogr. A 2011, 1218, 2676–2683

[2] Choma, I. M.; Jesionek, W. Effects-Directed Biological Detection: Bioautography. In Instrumental Thin-Layer Chromatography; Elsevier: Amsterdam, 2014

 JLU Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany, This email address is being protected from spambots. You need JavaScript enabled to view it.

On the one hand, questions such as “Which compounds of the thousands of compounds in a complex sample are effective?” can hardly be solved with a rational effort. On the other hand, direct bioautography, which could answer such a question, led to diffuse zones and was not convincing analysts. Recently, it was shown that sharply-bounded bioactive zones were available after several hours of incubation, and in combination with targeted mass spectrometry, led to a streamlined methodology that is able to answer such effect-directed questions within 15 min per sample [1-3].

The first part of the streamlined hyphenated method is an effect-directed screening of up to 22 raw extracts in parallel. Thus, this part (HPTLC-UV/Vis/FLD-(bio)assay) can be described as a non-targeted, effect-directed detection of single or also coeluting effective compounds of the complex sample. The second part is a highly targeted characterization of the discovered effective compounds via the direct link to structure elucidating techniques (HPTLC-HRMS or NMR or ATR FTIR). The zone of interest is selectively eluted via a short orthogonal column into the HRMS or into a microvial for NMR or ATR FTIR recordings. Thus, information on effective compounds in a complex sample and their sum formulae can be obtained from a single chromatographic run.

Depending on the bioassay or enzymatic assay selected, for example, œstrogens, antibiotics, xanthine oxidase inhibitors, acetylcholinesterase inhibitors or tyrosinase inhibitors are discovered in complex samples. However, every technique has its limitation, and for oxidation-prone or volatile compounds, this streamlined methodology is limited in the information content. Nevertheless, it may serve as a survey on effect-directed components in complex samples. Benefits may result from the side-by-side sample comparison, the matrix-tolerance, the avoidance of carry over and of discrimination, the always fresh adsorbent, the comparatively low-tech workflow as well as the multifold evaluation of the separated sample saved on the plate.

Literature:

[1] G.E. Morlock, ACS Syposium Series 1185 (2013) 101-121.

[2] G.E. Morlock, I. Klingelhöfer, Anal. Chem. 86 (2014) 8289–8295.

[3] I. Klingelhöfer, G.E. Morlock, J. Chromatogr. A 1360 (2014) 288-295.

 ¹ School of Pharmacy and Applied Science, La Trobe Institute of Molecular Sciences, La Trobe University, Edwards Rd, Bendigo 3550, Australia

² Physiology Department, Medical School, Universiti Teknologi Mara, Selangor, Malaysia, This email address is being protected from spambots. You need JavaScript enabled to view it.

This study describes a simple high performance thin layer chromatographic (HPTLC) method for the quantification of apigenin, chamazulene, bisabolol and parthenolide in leaf and flower head extracts from feverfew, German chamomile and marigold and comparison of their free radical scavenging activities. Feverfew (Tanacetum parthenium) leaves have been traditionally used in the treatment of migraine with parthenolide being the main bioactive compound. However, due to similar flowers, feverfew is sometimes mistaken for the German chamomile (Matricaria recutita). Bisabolol and chamazulene are the main components in chamomile essential oil. Marigold (Calendula officinalis) was included in the study for comparison, as it belongs to the same Asteraceae or Compositae (commonly known as daisy) family.

Parthenolide was found to be present in all leaf extracts but was not detected in calendula flower extract. Chamazulene and bisabolol were found to be present in higher concentrations in chamomile and Calendula flowers. Apigenin was detected and quantified only in chamomile extracts. Antioxidant activity in sample extracts was compared by superimposing the chromatograms obtained after post chromatographic derivatization with DPPH free radical and post chromatographic derivatization with anisaldehyde. It was found that extracts from chamomile flower heads and leaves have the most prominent antioxidant activity, with bisabolol and chamazulene being the most effective antioxidants.