Speed　of　analysis　is　a　key　factor　in　liquid　chromatography.　One　possible　way　is　to　decrease　the　particle　size　and　thus　increase　the　pressure　(ultra　high　pressure　liquid　chromatography,　UHPLC).　For　the　separation　of　a　complex　mixture,　the　application　of　gradient　elution　techniques　is　expedient.　From　a　practical　point　of　view,　it　has　to　be　questioned　how　the　high　pressure　influences　the　separation　parameters　at　gradient　mode.　In　our　study　we　investigated　the　following　chromatographic　parameters:　peak　width　versus　temperature,　peak　width　versus　gradient　time,　retention　time　versus　temperature,　and　retention　factor　versus　flow　rate.　It　was　shown　that　even　in　sample　volume　overload　conditions　the　gradient　system　worked　in　a　reproducible　way;　the　maximum　possible　sample　volume　(20　μL)　was　injected　to　get　the　lowest　possible　limit　of　detection.　Increasing　the　flow　rate,　the　peak　width　increased　only　moderately,　and　the　same　was　found　when　the　temperature　was　decreased.　However,　at　60°C　the　signal　to　noise　ratio　decreased　in　a　small　compass　showing　the　thermal　disequilibrium　between　the　column　outlet　and　the　detector　cell,　which　might　have　caused　extra　noise.　Finally,　a　practical　separation　was　presented　in　which　the　UHPLC　can　be　applied　for　the　determination　of　N-acylhomoserine　lantones　(AHLs),　which　have　a　relatively　low　UV　absorption　at　low　wavelength.　The　detection　limit　was　low　enough　for　practical　applications　because　of　the　low　dispersion　due　to　a　small　diameter　stationary　phase　and　the　low　dead　volume　in　the　column.　The　UPLC　method　was　applied　as　a　first　screening　for　AHLs　and　the　results　were　confirmed　by　fourier　transformation　ion　cyclotron　resonance　mass　spectrometry　(FTICR-MS),　which　has　a　high　selectivity.　Copyright　©　Taylor　&　Francis　Group,　LLC.