Browsing by discipline "Analyyttinen kemia"

The literature part of this thesis contains the review of affinity chromatography using monolithic stationary supports in the separation and isolation of biomacromolecules, a technique known as affinity monolith chromatography (AMC). Affinity chromatography is a liquid separation technique operating on the principle of reversible binding of affinity ligands and target analytes. Experimentally, affinity chromatography involves the attachment of affinity ligands to the stationary support. By selecting appropriate ligands having high affinity and specificity towards the target, selective captures of analytes of interest are made possible, allowing their isolation from complex sample matrices. Subsequently, bound analyte species are released from the ligands by employing suitable elution solutions. In addition to the specificity, monolithic stationary phases offer a number of other benefits over conventional particulate supports, i.e., improved mass transfer characteristics, allowing convective rather than diffusional transport of analytes; and high permeability, permitting operations at high flow rates without suffering from backpressure. These benefits result in substantially reduced time requirements for isolation and separation while maintaining satisfactory separation efficiency. Different types of monolithic materials, including organic polymer-based monoliths (e.g., cryogels), inorganic monoliths (e.g., silica monoliths), and hybrid monoliths have been prepared and employed in AMC. A large range of affinity ligands, e.g., proteins, antibodies, immobilized metal ions, dye ligands, have been used with monolithic supports in different formats, and in different applications. The mentioned material-related topics, as well as recent applications of AMC, are discussed in detail in this review. The experimental part of this thesis deals with the isolation of lipoproteins, and low-density lipoprotein (LDL) in particular, from human blood plasma using a newly developed AMC technique. LDL, a globular and major lipid carrier in blood, is diagnostically a highly relevant subclass of lipoproteins due to its involvement in the genesis of atherosclerosis. The currently most frequently employed method for lipoprotein isolation from blood plasma is ultracentrifugation. However, this method suffers from drawbacks, such as being time-consuming, requiring expensive equipment, and the possible exchange of lipids and lipoprotein subclasses during sample processing. Therefore, the first goal was to develop a faster LDL isolation protocol, capable of yielding LDL with good functionality and purity. Thus, the first section reports on the isolation of low-density lipoprotein (LDL) from human blood plasma by employing affinity monolith chromatography method using Convective Interaction Media (CIM) monolithic disk columns as stationary supports. Specifically, anti-apoB100-monoclonal antibody (mAb) was immobilized onto a CIM monolithic disk, providing a suitable capture medium for LDL through its major apolipoprotein, apolipoprotein B100 (apoB100). Other lipoprotein classes, namely very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL), also carry apoB100 and thus may be captured. To discriminate against these lipoproteins, and to obtain LDL with satisfactory purity, an additional CIM monolithic column was immobilized with a glycosaminoglycan, namely chondroitin-6-sulfate (C6S), which also binds lipoproteins, albeit with different specificity and interactions. Both of these affinity media were evaluated for LDL binding either individually or in combination. The quality of the isolated LDL was confirmed with different characterization techniques, such as size exclusion chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), enzymatic cholesterol and triglyceride assays, and enzymatic-linked immunoassays (ELISAs) specific to apolipoprotein B100 and apolipoprotein E. The results from these multi-method characterizations confirmed the successful LDL isolation with good activity. The second section of the thesis was devoted to quartz crystal microbalance (QCM) biosensor studies of LDL samples isolated from different individuals by different methods (affinity chromatography and conventional ultracentrifugation). A QCM sensor chip immobilized with anti-apoB100 mAb was used and challenged series of different LDL concentrations. The resulting sensorgrams were analyzed with a new numerical algorithm, namely Adaptive Interaction Distribution Algorithm (AIDA), permitting the determination of the number of analyte-receptor binding sites and the underlying kinetics. It was found that the obtained rate constant distributions, and clustering of antibody-LDL complexes were almost identical for all LDL samples, irrespective of sources or isolation techniques. For all samples, a total of five major complex clusters were identified. The major contributions of the two dominating clusters may arise from specific, yet heterogeneous LDL interactions at the antibody binding sites, while the other three clusters observed reflect most likely nonspecific low-affinity interactions from various sources, such as mass transfer effects, and the use of a non-orienting ligand immobilization chemistry.

In the literature part an introduction to emerging organic contaminants is presented as well as the subcategory of endocrine disrupting chemicals, which includes steroid hormones. An overview of liquid chromatography trends in environmental analysis as well as mass spectrometry operational conditions are discussed. Review is focused on ionization techniques and tandem mass spectrometry functionalities reported on state of the art studies of steroidal compounds in environmental samples. Experimental part presents an extensive sample preparation method and a liquid chromatography – mass spectrometry (HPLC-MS) method that were developed for the determination of concentration of eleven steroidal compounds: 4-androstene-3,17-dione (A1), trans-androsterone (A2), Corticosterone (C1), Cortisone (C2), Estrone (E1), 17-β-estradiol (E2), Estriol (E3), 17-α-ethynil estradiol (EE2), Progesterone (P), 17-hydroxy progesterone (HP) and Testosterone (T). Separation efficiency and analysis time were compared for three HPLC columns with different stationary phase: Pentafluorophenyl (PFP), C8 and C18 monolithic. Three atmospheric pressure ionization (API) techniques were tested to compare their performance: Electrospray ionization (ESI), Atmospheric Pressure Chemical Ionization (APCI) and Atmospheric Pressure Photoionization (APPI). The proposed method included the best column choice coupled with the API technique, which presented an efficient ionization for most of the target analytes. Best methodology was applied to the analysis of effluent and influent samples from the wastewater treatment plant in Viikinmäki, Helsinki.

Uusien mittaustekniikoiden nopea kehittyminen tuo mukanaan yhä paremmat mahdollisuudet tutkia biologisten molekyylien välisiä vuorovaikutuksia. Erityisesti lääkkeiden kehityksessä on ensiarvoisen tärkeää saada tietoa lääkeaineen ja biologisten molekyylien välisistä vuorovaikutuksista, sillä esimerkiksi lääkeaineen sitoutuminen plasman proteiineihin vaikuttaa merkittävästi lääkeaineen imeytymiseen, leviämiseen, metaboliaan ja eliminaatioon kehossa. Tärkeää on myös saada tietoa proteiini-hiilihydraatti ja vasta-aine-antigeeni vuorovaikutuksista, sillä ne liittyvät biologisiin prosesseihin, kuten hormonien toimintaan ja tunnistamiseen sekä biologisten molekyylien varastointiin. Biologisten molekyylien analytiikka on haastavaa, koska niiden aktiivisuus ja ominaisuudet usein muuttuvat niitä käsiteltäessä. Näistä mainittakoon mm. entsyymin aktiivisuus, joka saattaa kadota ja proteiinien denaturoituminen. Biosensori on analyyttinen laite, jossa biologista tai biologisesti johdettua materiaalia on joko sidottu tai kokonaan integroitu fysikaalis-kemialliseen anturiin. Biosensorien kehittämisen tavoitteena on valmistaa systeemejä, joiden avulla voidaan tutkia soluja, solukalvoja ja niiden ympäristöä reaaliajassa. Tarkoitus on saada biologisista prosesseista luotettavaa tietoa, jota hyödynnetään ihmisten hyvinvoinnin parantamiseksi. Biosensorien pitäisi olla edullisia, kestäviä, luotettavia ja lääketieteelliseen tutkimukseen soveltuvia. Tämän pro gradu -tutkielman kirjallisessa osassa tarkastellaan kvartsikidemikrovaa'an (QCM) hyödyntämistä biosensorina. QCM on laite, joka mittaa elektrodiin liitetyn kvartsikiteen värähtelytaajuutta. Kun sensorin massa muuttuu, myös sen värähtelytaajuus muuttuu. Värähtelytaajuuden muutoksesta voidaan tehdä johtopäätöksiä elektrodin pinnalla tapahtuvista muutoksista. Itsemuodostuvien pintakerrosten (SAM) avulla sensoripintaa voidaan muokata biologisten molekyylien havaitsemiseksi. SAM-pintojen käyttö perustuu kullan ja tioliyhdisteiden väliseen voimakkaaseen ja spontaaniin vuorovaikutukseen. Tioliyhdisteet muodostavat kullan pinnalle tasaisen ja stabiilin molekyylikerroksen. Tämän kerroksen ominaisuuksia muokkaamalla voidaan vaikuttaa pinnan kemiallisiin ja fysikaalisiin ominaisuuksiin. Kirjallisessa osassa käsitellään QCM:n soveltuvuutta biosensoriksi immunoglobuliini E:n ja kloramfenikolin kvantitointiin, R- ja S-enantiomeerien erotukseen, solutukirankaan sitoutuvien lääkeaineiden tutkimiseen sekä seerumin vasta-ainemääritykseen. Tulokset ovat varsin lupaavia, vaikka QCM-laitteiston herkkyys ei vielä monissa sovelluksissa oikein riitä kvantitatiiviseen määritykseen. Myös sensoripinnan uudelleenkäyttö aiheuttaa vielä ongelmia. Pro gradu -tutkielman kokeellisessa osassa sovellettiin kapillaarielektrokromatografiaa (CEC) matalatiheyksisen lipoproteiinin (LDL) ominaisuuksien tutkimuksiin. CEC:ssä käytettävien 50 µm halkaisijan avoputkisilikakapillaarien sisäpinta päällystettiin LDL:llä, joka toimi stationaarifaasina. Kehitetyllä menetelmällä tutkittiin mm. LDL:ssa tapahtuvia muutoksia sokerikäsittelyn ja hapettumisen jälkeen. Tutkimuksissa hyödynnettiin elektro-osmoottista liikkuvuutta pinnassa tapahtuvien muutosten indikaattorina. CEC osoittautui hyväksi tekniikaksi biologisella materiaalilla helposti muokattavien kapillaaripintojen, lyhyiden analyysiaikojen ja vähäisten reagenssi- ja näytemäärien ansiosta.

The large variety of shapes, functions, and conformations of proteins explains the challenges in protein identification and separation in solution. Gel electrophoresis, and more specifically SDS-PAGE, is an established technique applied to large proteins. Capillary gel electrophoresis offers the advantage of miniaturization coupled with higher speed of analysis and sample throughput. Protein analysis in silica capillaries is affected by the presence of charges on the silica surface, causing absorption, reliability and repeatability issues. Electroosmotic flow may also contribute, requiring buffer additives such as sodium dodecyl sulphate and dynamic or permanent coatings on the capillary wall. Coatings employed in capillary zone electrophoresis can be neutral or charged. They include copolymers such as poly((1-vinylpyrrolidone)-co-(2-dimethylaminoethyl methacrylate)), polyacrylamide, diazoresin as a coupling agent to form covalently bound coatings with polyvinyl alcohol, carboxyl fullerene and graphene oxide, and proprietary coatings. Capillary gel electrophoresis offers the advantages of a sieving matrix to enhance the separation of large proteins with similar charge-to-mass ratio. Dilute and semi-dilute polymer solutions with or without self-coating properties can be employed, such as polyacrylamide, polydimethyl acrylamide, and hydrophilic cellulose derivatives such as hydroxypropyl cellulose and hydroxyethyl cellulose. UV detection is enhanced by stacking techniques such as field-amplified sample stacking and the addition of sodium chloride to the sample. The focus of experimental laboratory works for this thesis was the identification of BSA, the lipoproteins HDL and LDL, and an apolipoprotein ApoB-100 using capillary gel electrophoresis. The polymer solution employed was cross-linked polyacrylamide. Instrumental factors such as run voltage, injection type, presence of sieving matrix and SDS in the BGE, sample concentration and presence of NaCl in the sample were examined. Repeatability was an issue throughout the study caused by current instability, although SDS in the BGE and addition of NaCl to the sample prior to injection had a positive effect. Stability of the BGE and the sieving matrix, together with addition of NaCl to the sample could be explored further.

This study focuses on chemometric analysis of instrumental data that has been obtained from chemical analysis of plant extracts. Chemometric analysis applies statistical and mathematical tools on chemical data, aiming to find new information or classifying samples in categories defined by the analyst. Chemometric analysis is based on computational pattern recognition and reveals any features that studied samples may have in common. In the literature part of this study, chemometrics and relevant concepts closely related to it are first explained and four commonly used chemometric methods are introduced, namely principal component analysis, hierarchical cluster analysis, k nearest neighbors and soft independent modeling of class analogy. The text is written with emphasis on being easily understandable without prior knowledge on the subject. After introducing these concepts, the literature concerning metabolomic studies of plant extracts published in the recent ten years are reviewed. This literature commonly employs chemometrics, aiming to discover if two or more varieties of the same plant species have markedly differing metabolomes and whether they can be exploited to automatically recognize these varieties. Additionally, the chemometric approaches often attempt to discover what factors are causing the successful findings. The purpose of the literature survey is to concretely show how chemometrics can achieve these goals, and to learn what the most common ways to treat the analytical data prior to chemometric analysis are. The experimental part applies chemometric methods to study bean extracts of the Ricinus communis plant, aiming to reveal if seed extracts of a same plant variety can be observed being similar, but clearly different from extracts of other varieties. Such situation could be exploited to develop a method that automatically identifies unknown seeds of the plant. The experimental work consisted of extracting homogenized samples with dilute aqueous acid, analyzing the extracts by three different instrumental techniques (liquid chromatography with ultraviolet light detection, liquid chromatography-mass spectrometry, and proton nuclear magnetic resonance spectroscopy) and finally analyzing the instrumental data by chemometric methods. Chemometrics research suffers from nonexistent standard operating procedures, since there is no universal way to treat a sample or data derived from it. While the main steps are often same, the details of sample preparation and preprocessing of analytical data vary greatly and can have a significant impact on the outcome. Despite, the data preprocessing is often left partially or completely manifested. The experimental finding was that six varieties of Ricinus communis could be successfully discriminated by both principal component analysis and hierarchical cluster analysis, applied on chromatographic data, while the results for spectroscopic data were not successful. The results encourage continuing the research, but with more emphasis on peak alignment and further experimenting with the preprocessing of the spectroscopic data. Choosing different short segments of the original spectroscopic profile is suggested, to leave out excessive information that is not helpful in discriminating the plant varieties but could obscure the relevant information.

The purpose of this study is to describe the development of application of mass spectrometry for the structural analyses of non-coding ribonucleic acids during past decade. Mass spectrometric methods are compared of traditional gel electrophoretic methods, the characteristics of performance of mass spectrometric, analyses are studied and the future trends of mass spectrometry of ribonucleic acids are discussed. Non-coding ribonucleic acids are short polymeric biomolecules which are not translated to proteins, but which may affect the gene expression in all organisms. Regulatory ribonucleic acids act through transient interactions with key molecules in signal transduction pathways. Interactions are mediated through specific secondary and tertiary structures. Posttranscriptional modifications in the structures of molecules may introduce new properties to the organism, such as adaptation to environmental changes or development of resistance to antibiotics. In the scope of this study, the structural studies include i) determination of the sequence of nucleobases in the polymer chain, ii) characterisation and localisation of posttranscriptional modifications in nucleobases and in the backbone structure, iii) identification of ribonucleic acid-binding molecules and iv) probing of higher order structures in the ribonucleic acid molecule. Bacteria, archaea, viruses and HeLa cancer cells have been used as target organisms. Synthesised ribonucleic acids consisting of structural regions of interest have been frequently used. Electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI) have been used for ionisation of ribonucleic analytes. Ammonium acetate and 2-propanol are common solvents for ESI. Trihydroxyacetophenone is the optimal MALDI matrix for ionisation of ribonucleic acids and peptides. Ammonium salts are used in ESI buffers and MALDI matrices as additives to remove cation adducts. Reverse phase high performance liquid chromatography has been used for desalting and fractionation of analytes either off-line of on-line, coupled with ESI source. Triethylamine and triethylammonium bicarbonate are used as ion pair reagents almost exclusively. Fourier transform ion cyclotron resonance analyser using ESI coupled with liquid chromatography is the platform of choice for all forms of structural analyses. Time-of-flight (TOF) analyser using MALDI may offer sensitive, easy-to-use and economical solution for simple sequencing of longer oligonucleotides and analyses of analyte mixtures without prior fractionation. Special analysis software is used for computer-aided interpretation of mass spectra. With mass spectrometry, sequences of 20-30 nucleotides of length may be determined unambiguously. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Sequencing in conjunction with other structural studies enables accurate localisation and characterisation of posttranscriptional modifications and identification of nucleobases and amino acids at the sites of interaction. High throughput screening methods for RNA-binding ligands have been developed. Probing of the higher order structures has provided supportive data for computer-generated three dimensional models of viral pseudoknots. In conclusion. mass spectrometric methods are well suited for structural analyses of small species of ribonucleic acids, such as short non-coding ribonucleic acids in the molecular size region of 20-30 nucleotides. Structural information not attainable with other methods of analyses, such as nuclear magnetic resonance and X-ray crystallography, may be obtained with the use of mass spectrometry. Sequencing may be applied to quality control of short synthetic oligomers for analytical purposes. Ligand screening may be used in the search of possible new therapeutic agents. Demanding assay design and challenging interpretation of data requires multidisclipinary knowledge. The implement of mass spectrometry to structural studies of ribonucleic acids is probably most efficiently conducted in specialist groups consisting of researchers from various fields of science.

Short-chain aliphatic amines (SCAA) are present in multiple different matrices in the environment at low concentration levels. SCAA are considered to be environmentally relevant compounds due to their role as precursors in the formation of carcinogenic N-nitrosoamines in various matrices and new particle formation in the atmosphere. SCAA are characteristically highly volatile, polar, reactive and basic compounds. Consequently, the quantitative determination of SCAA tends to be rather challenging. In the literature part of this thesis, different analytical methods used for the determination of SCAA in environmental samples are reviewed. The typical approach for the analysis of SCAA has been the use of derivatization techniques. Derivatization converts SCAA into less polar and less volatile form, which enables the use of conventional separation techniques, such as gas chromatography (GC) and high-performance liquid chromatography (HPLC). However, the methods involving derivatization can be quite time consuming, require the usage of excess reagents and are mainly applicable for the analysis of primary and secondary SCAA. To reduce the amount of reagent and solvent consumption, microextraction techniques have been implemented as part of the derivatization methods. For the analysis of free SCAA, mainly ion chromatography (IC) and GC have been used. In recent years, also novel online mass spectrometry techniques have been used for the determination of free SCAA in atmospheric air. In the experimental part of this thesis, a novel solid-phase microextraction (SPME) device called SPME Arrow was used for the extraction of free SCAA. Different SPME Arrow sorbent materials were tested, including commercial and custom sorbents, extraction conditions were optimized and the performance of SPME Arrow was compared to conventional SPME fiber. The developed method was applied for the determination of SCAA in wastewater samples and atmospheric air samples. In general, the performance of the custom sorbent coated SPME Arrow was not adequate due to the deterioration of coating, although the preliminary results indicated possible selectivity towards dimethylamine. Considering the commercial sorbent coated SPME devices, the SPME Arrow was better than the SPME fiber in terms of limit of quantification and performance in real sample analysis. When the SPME Arrow was used for wastewater sample analysis, no matrix interferences were observed, opposite to the results obtained with the SPME fiber. In addition, the SPME Arrow could be used for the determination of SCAA in atmospheric air samples following prior preconcentration by using denuder for sampling.

We humans utilise many kinds of chemicals, some of them are safe to use and some of them are dangerous to use. There are chemicals that fall into grey area in the terms of safety. Surfactants are one of them. They are used abundantly and they find their ways to the environment. It is an established fact that surfactants can more or less hinder normal functions of cells, and in the worst cases can cause cell deaths. Despite of this, it is not completely understood what harm surfactants can do to the living organisms in the environment. We live and work in houses that are cleaned with washing chemicals and surfactants. Recently, surfactants were supposed to exist in indoor air, and new studies prove this hypothesis. Literature explains that there might be the possibility that surfactants can adsorb into aerosols. However, analysis methods capable to be used directly for determination of surfactants in aerosol condensate samples are not available. In this M.Sc. thesis a new surfactant determination method was developed with capillary electrophoresis using UV detection and tetraborate complex formation. First surfactant determination methods, found from the literature for environmental samples were reviewed and described in this M.Sc. thesis. Then their suitability for experimental studies was evaluated. Among many options, capillary electrophoresis coupled with ultraviolet detection was selected. The method was developed for determination of didecyldimethylammonium chloride (DDAC) and polyethylene glycol monoalkyl ether (Genapol X-80), which are representatives of cationic and nonionic surfactans, respectively, and represent the surfactants in cleaning chemicals. In the experimental work method development was focused on composition of the electolyte solutions, since they played an important roles in separation and sensitivity of the analytes. First Tricine was selected for electrolyte, because it provided the best responses in the preliminary tests. However, in the later studies it, unfortunately, proved to be unsuitable for the determination of cationic and nonionic surfactants. Therefore, in accordance with published literature tetraborate electrolyte was chosen. As application studies, we demonstrated that the studied surfactants are present in water vapour by analysing seperately DDAC and Genapol X-80 in collected water condensates by laboratory scale piloting tests.The developed method was also applied to authentic samples of indoor water condensates and washing solutions that were collected from two elementary schools with air quality issues. Surfactants were detected in these samples too.

Paralytic shellfish poisoning toxins belong to a group of marine biotoxins that can cause severe food poisoning. The marine biotoxins have highly varying properties, such as molecular weight, solubility and toxicity. They accumulate into shellfish during harmful algal blooms. The global fish industry monitors sea food prior to releasing them to the market to ensure the safety of consumers, and permitted levels of marine biotoxins are regulated worldwide. Possible bioterrorism use of marine biotoxins is a concern to the governments due to their high toxicity and availability. The main emphasis in this thesis is on paralytic shellfish poisoning toxins, saxitoxin and its analogues. Saxitoxin is listed under the Chemical Weapons Convention. The paralytic shellfish poisoning toxin analogues share physico-chemical properties such as solubility, but they differ highly from each other in toxicity. The most toxic analogues of paralytic shellfish poisoning toxins are the Saxitoxin and Neosaxitoxin. The toxicity of the paralytic shellfish poisoning toxins is due to the blocking of the voltage gated sodium channels, which is a reversible process. The symptoms of paralytic shellfish poisoning can be numbness, weakness and even paralysis, which can lead to respiratory failure. The paralytic shellfish poisoning can be lethal and there is no antidote. A new liquid chromatography- tandem mass spectrometric method using multiple reaction monitoring was developed. Several different hydrophilic interaction liquid chromatography type analysis columns were compared. A liquid chromatography- high resolution mass spectrometric analysis methods using full scan and product ion scan were developed for several of the paralytic shellfish poisoning toxin analogues. The limit of detection and limit of quantification for Saxitoxin analyzed with the high resolution mass spectrometry method were 0.2 ng/ml and 0.7 ng/ml respectively. In average the detection and quantification limits obtained with the high resolution mass spectrometry were around ten times better than the values obtained using triple quadrupole mass spectrometry. New sample preparation methods were developed for four different matrices (mussel, urine, milk and juice) applying solid phase extraction as the primary sample clean-up technique to be used in proficiency tests. Proficiency test samples analyzed during this thesis contained mussel, urine and unknown saxitoxin samples. The proficiency test samples were analyzed using three different chromatography-based analysis techniques to determine the presence of paralytic shellfish poisoning toxins. A comparison was done between the developed triple quadrupole mass spectrometric method, high resolution mass spectrometric method and a standardized fluorescence detection method.

Denna avhandling behandlar elektrokinetisk kapillärkromatografi (EKC) och omfattar en litteraturöversikt och en experimentell studie. Litteraturöversikten består av en inledande teoretisk genomgång av principerna för EKC. Den följs av en presentation av litteraturen rörande tillämpning av EKC vid analys av lokalbedövningsmedel. Micellär elektrokinetisk kapillärkromatografi (MEKC) är den äldsta versionen av EKC och hittills den mest använda för separation av lokalbedövningsmedel. Under de senaste åren har dock speciellt användningen av vesiklar och lipiddispersioner samt mikroemulsioner som pseudostationär fas ökat. I den experimentella delen av arbetet användes liposom elektrokinetisk kapillärkromatografi (LEKC) för att studera växelverkningar mellan bioimiterande membraner och sex lokalbedövningsmedel och ett typiskt konserveringsmedel. De bioimiterande membranerna bestod av liposomer uppbyggda av 1-palmitoyl-2-oleyl-sn-glycero-3-fosfatidylkolin (POPC) och 1-palmitoyl-2-oleyl-sn-glycero-3-[fosfo-rac-(1-glycerol)] (POPG), med eller utan kolesterol. Retentionsfaktorer och fördelningskonstanter bestämdes för samtliga analyter. Liposomernas mobilitet, vilken behövs för beräkning av analyternas retentionsfaktorer, bestämdes genom en iterativ procedur som innebar bestämning av retentionsfaktorer för en homologisk serie alkylbenzoater. Analyternas fördelning i liposomer jämfördes med litteraturvärden för fördelningen i oktanol/vatten-system. Temperaturens inverkan på växelverkningarna undersöktes genom att utföra separationer vid 25°C, 37°C och 42°C. Studierna visade att analyternas fördelning i liposomerna minskar då kolesterol tillsätts till liposomerna. Temperaturförändringen hade en smärre, osystematisk inverkan på växelverkningarna. Korrelationen mellan de experimentellt bestämda fördelningskonstanterna i liposom/vatten-system och litteraturvärden för fördelningen i oktanol/vatten-system är relativt svag.