Interfacial Tension Analysis of Oligo(ethylene glycol)-Terminated Self-Assembled Monolayers and Their Resistance to Bacterial Attachment

The fouling resistance of oligo(ethylene glycol) (OEG)-terminated self-assembled monolayers (SAMs) of alkanethiolates on gold has been well established. Although hydration of the OEG chains seems key to OEG-SAM resistance to macromolecular adsorption and cellular attachment, the details of how hydration prevents biofouling have been inferred largely through computational methods. Because OEG-SAMs of different lengths exhibit differing degrees of fouling resistance, the interactions between water and OEG-SAMs leading to fouling resistance can be deduced by comparing the properties of fouling and nonfouling OEG-SAMs. While all OEG-SAMs had similar water contact angles, contact angles taken with glycerol were able to individuate between different OEG-SAMs and between fouling and nonfouling OEG-SAMs. Subsequent estimation of surface and interfacial tension using a colloidal model showed that nonfouling surfaces are associated with an increased negative interfacial tension between those OEG-SAMs that resisted attachment and water. Further analysis of this interfacial tension experimentally confirmed current mathematical models that cite OEG-water hydrogen-bond formation as a driving force behind short-term fouling resistance. Finally, we found a correlation between solid-water interfacial tension and packing density and molecular density of ethylene glycol.     

Factors that determine the protein resistance of oligoether self-assembled monolayers --internal hydrophilicity,terminal hydrophilicity,and lateral packing density

Protein resistance of oligoether self-assembled monolayers (SAMs) on gold and silver surfaces has been investigated systematically to elucidate structural factors that determine whether a SAM will be able to resist protein adsorption. Oligo(ethylene glycol) (OEG)-, oligo(propylene glycol)-, and oligo(trimethylene glycol)-terminated alkanethiols with different chain lengths and alkyl termination were synthesized as monolayer constituents. The packing density and chemical composition of the SAMs were examined by XPS spectroscopy; the terminal hydrophilicity was characterized by contact angle measurements. IRRAS spectroscopy gave information about the chain conformation of specific monolayers; the amount of adsorbed protein as compared to alkanethiol monolayers was determined by ellipsometry. We found several factors that in combination or by themselves suppress the protein resistance of oligoether monolayers. Monolayers with a hydrophobic interior, such as those containing oligo(propylene glycol), show no protein resistance. The lateral compression of oligo(ethylene glycol) monolayers on silver generates more highly ordered monolayers and may cause decreased protein resistance, but does not necessarily lead to an all-trans chain conformation of the OEG moieties. Water contact angles higher than 70 degrees on gold or 65 degrees on silver reduce full protein resistance. We conclude that both internal and terminal hydrophilicity favor the protein resistance of an oligoether monolayer. It is suggested that the penetration of water molecules in the interior of the SAM is a necessary prerequisite for protein resistance. We discuss and summarize the various factors which are critical for the functionality of "inert" organic films.     

Alpha,omega-functionalized poly-N-isopropylacrylamides: controlling the surface activity for vesicle adsorption by temperature

The synthesis of alpha,omega-end-functionalized copolymers of N-isopropylacrylamide and N-(3-dimethylaminopropyl)acrylamide was performed. Monomer ratios of 100:0, 96:4, and 81:19 were investigated. The lower critical solution temperature (LCST) of these polymers was determined by cloud-point measurements and by microcalorimetric measurements. The LCST increased from 32 over 37 to 47 degrees C as the hydrophobicity increased with increasing amount of comonomer N-(3-dimethylaminopropyl)acrylamide. The polymers could successfully be adsorbed onto gold surfaces. Finally, vesicle adsorption onto these self-assembled polymer films on flat gold surfaces was investigated as the vesicle solution temperature was varied. It could be observed that vesicle adsorption was hindered as long as the temperature of the vesicle solution was above the LCST of the polymer. As soon as it dropped below the LCST the vesicle adsorption process was initiated.     

Molecular simulation study of water interactions with oligo (ethylene glycol)-terminated alkanethiol self-assembled monolayers

Molecular simulations were performed to study a system consisting of protein (e.g., lysozyme) and self-assembled monolayers (SAMs) terminating with different chemical groups in the presence of explicit water molecules and ions. Mixed SAMs of oligo (ethylene glycol) [S(CH2)4(OCH2CH2)4OH, (OEG)] and hydroxyl-terminated SAMs [S(CH2)4OH] with a mole fraction of OEG at chiOEG = 0.2, 0.5, 0.8, and 1.0 were used in this study. In addition, methyl-terminated SAMs [S(CH2)11CH3] were also studied for comparison. The structural and dynamic behavior of hydration water, the flexibility and conformation state of SAMs, and the orientation and conformation of protein were examined. Simulation results were compared with those of experiments. It appears that there is a correlation between OEG surface resistance to protein adsorption and the surface density of OEG chains, which leads to a large number of tightly bound water molecules around OEG chains and the rapid mobility of hydrated SAM chains.     

Protein adsorption on oligo(ethylene glycol)-terminated alkanethiolate self-assembled monolayers: The molecular basis for nonfouling behavior

A study of protein resistance of oligo(ethylene glycol) (OEG), HS(CH2)11(OCH2CH2)nOH (n = 2, 4, and 6), self-assembled monolayers (SAMs) on Au(111) surfaces is presented here. Hydroxyl-terminated OEG-SAMs are chosen to avoid the hydrophobic effect observed with methyl-terminated OEG-SAMs, particularly at high packing densities. The structure of the OEG-SAM surfaces is controlled by adjusting the assembly solvent. These SAMs were characterized by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). Protein adsorption on these surfaces was investigated by surface plasmon resonance (SPR). OEG-SAMs assembled from mixed ethanol and water solutions show higher packing density on gold than those from pure ethanol solution. For EG2OH- and EG4OH-SAMs, proteins (i.e., fibrinogen and lysozyme) adsorb more on the densely packed SAMs prepared from mixed ethanol and water solutions, while EG6OH-SAMs generally resist protein adsorption regardless of the assembly solvent used.     

An in situ study of the adsorption behavior of functionalized particles on self-assembled monolayers via different chemical interactions

The formation of particle monolayers by convective assembly was studied in situ with three different kinds of particle-surface interactions: adsorption onto native surfaces, with additional electrostatic interactions, and with supramolecular host-guest interactions. In the first case carboxylate-functionalized polystyrene (PS-COOH) particles were assembled onto native silicon oxide surfaces, in the second PS-COOH onto protonated amino-functionalized (NH3+) self-assembled monolayers (SAMs), and in the third beta-CD-functionalized polystyrene (PS-CD) particles onto beta-CD SAMs with pre-adsorbed ferrocenyl-functionalized dendrimers. The adsorption and desorption behaviors of particles onto and from these surfaces were observed in situ on a horizontal deposition setup, and the packing density and order of the adsorbed particle lattices were compared. The desorption behavior of particles from surfaces was evaluated by reducing the temperature below the dew point, thus initiating water condensation. Particle lattices on native oxide surfaces formed the best hexagonal close packed (hcp) order and could be easily desorbed by reducing the temperature to below the dew point. The electrostatically modified assembly resulted in densely packed, but disordered particle lattices. The specificity and selectivity of the supramolecular assembly process were optimized by the use of ferrocenyl-functionalized dendrimers of low generation and by the introduction of competitive interaction by native beta-CD molecules during the assembly. The fine-tuned supramolecularly formed particle lattices were nearly hcp packed. Both electrostatically and supramolecularly formed lattices of particles were strongly attached to the surfaces and could not be removed by condensation.     

Globotriose- and oligo(ethylene glycol)-terminated self-assembled monolayers: surface forces, wetting, and surfactant adsorption

A set of oligo(ethylene glycol)-terminated and globotriose-terminated self-assembled monolayers (SAMs) has been prepared on gold substrates. Such model surfaces are well defined and have good stability due to the strong binding of thiols and disulfides to the gold substrate. They are thus very suitable for addressing questions related to effects of surface composition on wetting properties, surface interactions, and surfactant adsorption. These issues are addressed in this report. Accurate wetting tension measurements have been performed as a function of temperature using the Wilhelmy plate technique. The results show that the nonpolar character of oligo(ethylene glycol)-terminated SAMs increases slightly but significantly with temperature in the range 20-55 degrees C. On the other hand, globotriose-terminated SAMs are fully wetted by water at room temperature. Surface forces measurements have been performed and demonstrated that the interactions between oligo(ethylene glycol)-terminated SAMs are purely repulsive and similar to those determined between adsorbed surfactant layers with the same terminal headgroup. On the other hand, the interactions between globotriose-terminated SAMs include a short-range attractive force component that is strongly affected by the packing density in the layer. In some cases it is found that the attractive force component increases with contact time. Both these observations are rationalized by an orientation- and conformation-dependent interaction between globotriose headgroups, and it is suggested that hydrogen-bond formation, directly or via bridging water molecules, is the molecular origin of these effects.     

Molecular dynamics simulations of peptide-surface interactions

Proteins, which are bioactive molecules, adsorb on implants placed in the body through complex and poorly understood mechanisms and directly influence biocompatibility. Molecular dynamics modeling using empirical force fields provides one of the most direct methods of theoretically analyzing the behavior of complex molecular systems and is well-suited for the simulation of protein adsorption behavior. To accurately simulate protein adsorption behavior, a force field must correctly represent the thermodynamic driving forces that govern peptide residue-surface interactions. However, since existing force fields were developed without specific consideration of protein-surface interactions, they may not accurately represent this type of molecular behavior. To address this concern, we developed a host-guest peptide adsorption model in the form of a G(4)-X-G(4) peptide (G is glycine, X is a variable residue) to enable determination of the contributions to adsorption free energy of different X residues when adsorbed to functionalized Au-alkanethiol self-assembled monolayers (SAMs). We have previously reported experimental results using surface plasmon resonance (SPR) spectroscopy to measure the free energy of peptide adsorption for this peptide model with X = G and K (lysine) on OH and COOH functionalized SAMs. The objectives of the present research were the development and assessment of methods to calculate adsorption free energy using molecular dynamics simulations with the GROMACS force field for these same peptide adsorption systems, with an oligoethylene oxide (OEG) functionalized SAM surface also being considered. By comparing simulation results to the experimental results, the accuracy of the selected force field to represent the behavior of these molecular systems can be evaluated. From our simulations, the G(4)-G-G(4) and G(4)-K-G(4) peptides showed minimal to no adsorption to the OH SAM surfaces and the G(4)-K-G(4) showed strong adsorption to the COOH SAM surface, which is in agreement with our SPR experiments. Contrary to our experimental results, however, the simulations predicted a relatively strong adsorption of G(4)-G-G(4) peptide to the COOH SAM surface. In addition, both peptides were unexpectedly predicted to adsorb to the OEG surface. These findings demonstrate the need for GROMACS force field parameters to be rebalanced for the simulation of peptide adsorption behavior on SAM surfaces. The developed methods provide a direct means of assessing, modifying, and validating force field performance for the simulation of peptide and protein adsorption to surfaces, without which little confidence can be placed in the simulation results that are generated with these types of systems.     

pH-Dependent force spectroscopy of tri(ethylene glycol)- and methyl-terminated self-assembled monolayers adsorbed on gold

Self-assembled monolayers (SAMs) of methoxy-tri(ethylene glycol)- (EG(3)-OMe) and methyl-terminated alkanethiols (C(16)) adsorbed on polycrystalline gold were investigated by chemical force spectroscopy. Measurements were performed in aqueous electrolyte solutions depending on ionic strength and pH value. Charged and hydrophobic tips were employed as probes to mimic local patches of proteins and to study the interaction at the organic/liquid interface in detail. Force-distance curves reveal information about the origin of the observed interaction and the underlying mechanisms. The measurements confirm an effective negative surface charge to be present at the oligo(ethylene glycol) (OEG) and the methyl interface and suggest that the charges are due to the adsorption of hydroxyl ions from aqueous solution. pH-dependent measurements further support the robustness of the established charge associated with the OEG films. Its sign does not change over the whole range of investigated values between pH approximately 3.5 and approximately 10. In contrast, the hydrophobic self-assembled hexadecanethiol films on gold show an isoelectric point (IEP) around pH 4. While the mechanism of charge establishment appears to be similar for both SA films, the strength of hydrogen bonding to interfacial water, which acts as a template for hydroxyl ion adsorption, is likely to be responsible for the observed difference.     

Adsorption of poly(N-isopropylacrylamide) on glass substrata

The adsorption of poly(N-isopropylacrylamide) (PNIPAAM), a well known thermosensitive polymer, on glass was investigated by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The polymer was dissolved in water at low (0.02 g/L) and high (2 g/L) concentration and the tested temperatures were below (25 degrees C) and above (50 degrees C) the lower critical solubility temperature (LCST). Whatever the conditions, a smooth layer of adsorbed molecules spread along the surface was observed. The thickness was about twice higher for high concentration compared to low concentration. The cohesion in the adsorbed layer, as revealed by scraping tests performed by AFM, was higher above the LCST than below the LCST. On top of this adsorbed layer, single-chain coils, globules, or aggregates were present, depending on concentration and temperature. The observation of these additional adsorbed entities was poorly reproducible, presumably due to the lack of shear control upon rinsing. These results emphasize the importance of the characterization of surface morphology to interpret amounts of adsorbed polymers.     

Surface structure and interface dynamics of alkanethiol self-assembled monolayers on Au(111)

Scanning tunneling microscopy (STM) and high-resolution electron energy loss spectroscopy (HREELS) were used to examine the structural transitions and interface dynamics of octanethiol (OT) self-assembled monolayers (SAMs) caused by long-term storage or annealing at an elevated temperature. We found that the structural transitions of OT SAMs from the c(4 x 2) superlattice to the (6 x square root 3) superlattice resulting from long-term storage were caused by both the dynamic movement of the adsorbed sulfur atoms on several adsorption sites of the Au(111) surface and the change of molecular orientation in the ordered layer. Moreover, it was found that the chemical structure of the sulfur headgroups does not change from monomer to dimer by the temporal change of SAMs at room temperature. Contrary to the results of the long-term-stored SAMs, it was found that the annealing process did not modify either the interfacial or chemical structures of the sulfur headgroups or the two-dimensional c(4 x 2) domain structure. Our results will be very useful for a better understanding of the interface dynamics and stability of sulfur atoms in alkanethiol SAMs on Au(111) surfaces.     

Molecular orientation in helical and all-trans oligo(ethylene glycol)-terminated assemblies on gold: results of ab initio modeling

The structural properties of self-assembled monolayers (SAMs) of oligo(ethylene glycol) (OEG)-terminated and amide-containing alkanethiols (HS(CH(2))(15)CONH(CH(2)CH(2)O)(6)H and related molecules with shorter alkyl or OEG portions) on gold are addressed. Optimized geometry of the molecular constituents, characteristic vibration frequencies, and transition dipole moments are obtained using density-functional theory methods with gradient corrections. These data are used to simulate IR reflection-absorption (RA) spectra associated with different OEG conformations. It is shown that the positions and relative intensities of all characteristic peaks in the fingerprint region are accurately reproduced by the model spectra within a narrow range of the tilt and rotation angles of the alkyl plane, which turns out to be nearly the same for the helical and all-trans OEG conformations. In contrast, the tilt of the OEG axis changes considerably under conformational transition from helical to all-trans OEG. By means of ab initio modeling, we also clarify other details of the molecular structure and orientation, including lateral hydrogen bonding, the latter of which is readily possessed by the SAMs in focus. These results are crucial for understanding phase and folding characteristics of OEG SAMs and other complex molecular assemblies. They are also expected to contribute to an improved understanding of the interaction with water, ions, and ultimately biological macromolecules.     

Adsorption/desorption of mono- and diblock copolymers on surfaces using specific hydrogen bonding interactions

Diblock copolymers containing recognition units designed to participate in specific three-point hydrogen bonding were adsorbed onto modified gold surfaces. Self-assembled monolayers (SAMs) containing complementary recognition units were used to direct the adsorption process. The polymer-modified surfaces obtained were characterized using X-ray photoelectron spectroscopy, water contact angle, and ellipsometry. The role of individual block lengths on the adsorption process was followed by observing frequency changes of thymine-SAM-modified quartz crystal microbalance chips during adsorption of diamidopyridine-functionalized polymers from a nonpolar solvent. The renewable nature of these recognition unit functionalized surfaces was demonstrated by reversible binding of polymers. Adsorption onto fresh surfaces, followed by desorption and subsequent readsorption of monoblock and diblock copolymers was also investigated.     

Ice nucleation and phase behavior on oligo(ethylene glycol) and hydroxyl self-assembled monolayers: simulations and experiments

The nucleation and phase behavior of ultrathin D2O-ice overlayers have been studied on oligo(ethylene glycol) (OEG)-terminated and hydroxyl self-assembled monolayers (SAMs) at low temperatures in ultrahigh vacuum. Infrared reflection-absorption spectroscopy (IRAS) is used to characterize the ice overlayers, the SAMs, and the interactions occurring between the ice and the SAM surfaces. Spectral simulations, based on optical models in conjunction with Maxwell Garnett effective medium theory, point out the importance of including voids in the modeling of the ice structures, with void fractions reaching 60% in some overlayers. The kinetics of the phase transition from amorphous-like to crystalline-like ice upon isothermal annealing at 140 K is found to depend on the conformational state of the supporting OEG SAM surface. The rate is fast on the helical OEG SAMs and slow on the corresponding all-trans SAMs. This difference in kinetics is most likely due to a pronounced D2O interpenetration and binding to the all-trans segments of the ethylene glycol portion of the SAM. No such penetration and binding was observed on the helical OEG SAM.     

Synthesis and self-assembly of galactose-terminated alkanethiols and their ability to resist proteins

The synthesis of two galactose-terminated alkanethiols with the structural formula X-OC2H5NHCO(CH2)15SH (X = 2,3,4,6-tetra-O-methyl-beta-D-Gal or beta-D-Gal) is described. Single-component and mixed self-assembled monolayers (SAMs) of the methylated and nonmethylated compounds were prepared on gold and subsequently characterized with ellipsometry, contact angle goniometry, and infrared reflection-absorption spectroscopy. Studies of the irreversible protein adsorption onto the SAMs using ex-situ ellipsometry revealed very low levels of fibrinogen and lysozyme adsorption onto mixed SAMs displaying advancing water contact angles between 24 degrees and 45 degrees and below 45 degrees , respectively. A monomethylated compound (X = 6-O-methyl-beta-D-Gal) was also synthesized and assembled on gold. This particular compound was found to possess wettability properties corresponding to the low adsorption regime of the mixed SAMs, and the results from the same set of fibrinogen and lysozyme adsorption experiments showed very low levels of protein adsorption. Our findings suggest that the protein rejecting properties rely on a fine balance between the surface energy and/or hydrogen bond donating/accepting properties of the SAM surface.     

Hydrogel microspheres III. Temperature-dependent adsorption of proteins on poly-N-isopropylacrylamide hydrogel microspheres

Precipitation polymerization of N-isopropylacrylamide (NIPAM) with methylenebisacrylamide (MBAAm) in water at 70°C gave thermosensitive hydrogel microspheres. The adsorbability of proteins on the poly-NIPAM microspheres was found to depend on temperature. Below the lower critical solution temperature (LCST) of poly-NIPAM in an aqueous medium, that is, around 32°C, the microspheres hold a large amount of water inside and their surface is hydrophilic enough to suppress the adsorption of proteins. On the contrary, above 32°C, the micropheres deswell and their surface becomes hydrophobic and, consequently, susceptible to adsorption of a large amount of proteins. Proteins once adsorbed on the microspheres at a high temperature could be desorbed more or less by lowering the temperature to below 32°C. The extent of desorption at low temperatures was found to depend on the incubation time for adsorption at high temperatures.     

Formation of ordered self-assembled monolayers by adsorption of octylthiocyanates on Au(111)

Self-assembled monolayers (SAMs) were formed by the spontaneous adsorption of octythiocyanate (OTC) on Au(111) using both solution and ambient-pressure vapor deposition methods at room temperature and 50 degrees C. The surface structures and adsorption characteristics of the OTC SAMs on Au(111) were characterized by scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS). The STM observation showed that OTC SAMs formed in solution at room temperature have unique surface structures including the formation of ordered and disordered domains, vacancy islands, and structural defects. Moreover, we revealed for the first time that the adsorption of OTC on Au(111) in solution at 50 degrees C led to the formation of SAMs containing small ordered domains, whereas the SAMs formed by vapor deposition at 50 degrees C had long-range ordered domains, which can be described as (radical3 x 2 radical19)R5 degrees structures. XPS measurements of the peaks in the S 2p and N 1s regions for the OTC SAMs showed that vapor deposition is the more effective method as compared to solution deposition for obtaining high-quality SAMs by adsorption of OTC on gold. The results obtained will be very useful in understanding the SAM formation of organic thiocyanates on gold surfaces.     

Protein-resistant self-assembled monolayers on gold with latent aldehyde functions

In the present study, oligo(ethylene glycol) (OEG)-linked alkanethiols were synthesized which carry a vicinal diol on one end of the OEG chain. After self-assembled monolayer (SAM) formation on gold, the vicinal diols were converted into aldehyde functions by exposure to aqueous NaIO4, as previously used for SAMs with OEG chains buried in the center of the SAM [Jang et al. Nano Lett. 2003, 3, 691-694]. Mixed SAMs with latent aldehydes on 5% of the OEG termini showed high protein resistance, which greatly slowed the kinetics of protein coupling on the time scale of minutes. Small bioligands (such as biocytin hydrazide) or small heterobifunctional crosslinkers (maleimidopropionyl hydrazide, pyridyldithiopropionyl hydrazide) with hydrazide functions were efficiently bound to the aldehyde functions on the SAM, providing for specific capture of streptavidin or for fast covalent binding of proteins with free thiols or maleimide functions, respectively. In conclusion, OEG-terminated SAMs with latent aldehydes serve as protein-resistant sensor surfaces which are easily functionalized with small ligands or with heterobifunctional crosslinkers to which the bait molecule is attached in a subsequent step.     

Mixed self-assembled monolayers (SAMs) consisting of methoxy-tri(ethylene glycol)-terminated and alkyl-terminated dimethylchlorosilanes control the non-specific adsorption of proteins at oxidic surfaces

Monolayers from the newly synthesized compound methoxy-tri(ethylene glycol)-undecenyldimethylchlorosilane (CH3O(CH2CH2O)3(CH2)11Si(CH3)2Cl, MeO(EG)3C11DMS) and dodecyldimethylchlorosilane (DDMS), both pure and mixed, were prepared by self-assembly from organic solution in the presence of an organic base. The films obtained were characterized by advancing and receding contact angle measurements and ellipsometry to confirm the formation of self-assembled monolayers (SAMs). The resulting data on the covalently attached dimethylsilanes were compared to known oligo(ethylene glycol) (OEG)-terminated SAM systems based on terminal alkenes, thiolates or trihydrolyzable silanes. The composition of the mixed SAMs was found to depend directly and linearly on the composition of the silanization solution. Enhanced protein repellent properties were found for the SAMs using a variety of proteins, including the Ras Binding Domain (RBD), a protein with high relevance for cancer diagnostics. Roughly a RBD protein monolayer amount was adsorbed to silicon oxide surfaces silanized with DDMS or non-silanized silicon wafers, and in contrast, no RBD was adsorbed to surfaces silanized with MeO(EG)3C11DMS or to mixed monolayers consisting of DDMS and MeO(EG)3C11DMS if the content of OEG-silane overcame a critical content of X(EG) approximately 0.9.     

Effects of graft densities and chain lengths on separation of bioactive compounds by nanolayered thermoresponsive polymer brush surfaces

We have prepared various poly(N-isopropylacrylamide) (PIPAAm)-grafted silica bead surfaces through surface-initiated atom transfer radical polymerization (ATRP) by changing graft densities and brush chain lengths. The prepared surfaces were characterized by chromatographic analysis using the modified silica beads as chromatographic stationary phases. ATRP initiator (2-(m,p-chloromethylphenyl)ethyltrichlorosilane) density on silica bead surfaces was modulated by changing the feed composition of the self-assembled monolayers (SAMs) of mixed silane coupling agents consisting of ATRP initiator and phenethyltrichlorosilane on the surfaces. IPAAm was then polymerized on SAM-modified silica bead surfaces by ATRP in 2-propanol at 25 degrees C. The chain length of the grafted PIPAAm was controlled by simply changing the ATRP reaction time at constant catalyst concentration. The thermoresponsive surface properties of the PIPAAm-grafted silica beads were investigated by temperature-dependent elution behavior of hydrophobic steroids from the surfaces using Milli-Q water as a mobile phase. On the surfaces grafted with shorter PIPAAm chains, longer retention times for steroids were observed on sparsely grafted PIPAAm surfaces compared to dense PIPAAm brushes at low temperature, because of hydrophobic interactions between the exposed phenethyl groups of SAMs on silica surfaces and steroid molecules. Retention times for steroids on dilute PIPAAm chain columns decreased with temperature similarly to conventional reverse-phase chromatographic modes on octadecyl columns. This effect was due to limited interaction of solutes with the PIPAAm-grafted surfaces. Retention times for steroids on dilute PIPAAm brush surfaces with longer PIPAAm chains became greater above the PIPAAm transition temperature. At low-temperature regions, hydrated and expanded PIPAAm at low temperatures prevented hydrophobic interactions between the phenethyl group of SAMs on the silica bead surfaces and steroid molecules. Retention times for steroids on a dense PIPAAm brush column increased with temperature since solvated polymer segments within the dense brush layer undergo dehydration over a broad range of temperatures. In conclusion, PIPAAm graft density has a crucial influence on the elution behavior of steroids because of the interaction of analytes with silica bead interfaces, and because of the characteristic dehydration of PIPAAm in dense-pack brush surfaces.