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HPLC applikationer för wide pore Kromasil
2004 (Swedish)Independent thesis Basic level (professional degree)Student thesis
Abstract [en]

Kromasil is a packingmaterial made of silika that is used for packing HPLC columns. A new product with larger pores (300Å) has been developed for separating larger molecules like proteins. In this project this material has been examined and compared with different competitors. The competing materials are Jupiter, Vydac, Daiso, YMC and Polymer Lab. First, the quality of the stationary phase was examined on the different columns. When the efficiency of the columns was examined, Kromasil and YMC displayed the best efficiency. The hydrofobicity was also examined and this test showed that Kromasil, Jupiter and YMC was most hydrofobic. Jupiter was the column that could separate the basic analytes most efficiently. The ability to separate a mixture of different proteins was examined. Two mixtures of proteins was used. The first mixture contained ribonuclease A, insulin, cytocrome c, lysozyme and myoglobin. The second mixture contained oxytocin, bradykinin, angiotensin I, BSA and ovalbumin. The mobile phase was A: water/acetonitril/TFA (90/10/0.1, v/v/v) and B: water/acetonitrile/TFA (5/95/0.1, v/v/v). The first protein mix was examined using the gradient 20-60% ACN under 22 min and the second protein mix was examined using the gradient 20-70% ACN under 30 min. The separation was comparable for all columns. The proteins were eluted in the same order. The Kromasil column was tested at three different temperatures, 10, 20 and 35oC. The time for elution changed with the temperature. The Kromasil column was also examined using three different buffers. The buffers were 0.1 M Tris pH 7.4, 0.1 M NH4Ac pH 6 and (NH4)2SO4 pH 3.6. With the first protein mix only insulin eluted when the buffers with the pH 7.4 and pH 6 was used. When the buffer with the pH 3.6 was used insulin and cytocrome c eluted. With the second protein mix it was only oxytocin, bradykinin and angiotensin I that eluted. The dynamic capacity of the adsorption has been examined for the different columns. Two proteins were used, BSA and myoglobin. It was shown that the stationary phase could adsorb myoglobin better than BSA. For all columns the capacity of adsorption increased with a reduced flow. The Polymer Lab column had the highest capacity of adsorption but it was also most sensitive to the flow, which means that the mass transport was bad. Least sensitive to the flow was Jupiters column.

Place, publisher, year, edition, pages
2004. , 55 p.
Identifiers
URN: urn:nbn:se:kau:diva-52533Local ID: KEM-21OAI: oai:DiVA.org:kau-52533DiVA: diva2:1101060
Subject / course
Chemistry
Available from: 2017-05-29 Created: 2017-05-29

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