Ion Chromatography: Purification of Albumin Using CM-Cellulose Resin

ION CHROMATOGRAPHY 1
ION CHROMATOGRAPHY
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ION CHROMATOGRAPHY 2
Albumin analyte(1) is the most important component of the plasma proteins sample(1)
(Poltep, Tesena, Yingchutrakul, Taylor, & Wongtawan, 2018).In this experiment two different
methods are used for the albumin purification namely; carboxymethyl cellulose(CM) resin
exchange and Diethylamine cellulose exchange resin stationary phase(3) (Poltep, Tesena,
Yingchutrakul, Taylor, & Wongtawan, 2018). A suitable method for the albumin purification
was then chosen from the two. For this research, albumin purification using CM-cellulose resin
was found to be more beneficial, about 75% of the purified protein was found to be albumin in
the DEAE Cellulose resin while more than 90% of the plasma protein was albumin in the CM
method justifying CM to be the most beneficial method to be used (Poltep, Tesena,
Yingchutrakul, Taylor, & Wongtawan, 2018). Conductivity detector(6) was used in the research
since eluent was very mobile and was of good concentration.
Before the start of the purification process, qualitative calibration (7) was carried out
(Poltep, Tesena, Yingchutrakul, Taylor, & Wongtawan, 2018). The resin was washed with 3
fold of the primary buffer volume which is thee eluent (3)each containing 0.5ml of resin.0.5ml of
the tris buffer containing 0.1M Cl ions concentration was mixed with 2500 rpm suppressor to
create suspension,the type of ion echange used in this therefore is a suppressed IC method (2)
(Akbarzadehlaleh et al., 2016). 0.5 of the starting buffer was then added through gradient
elution(5),which is achieved when the buffer strength being increased by changing the
composition of the buffer and then mixed with the shaker for 5minutes (Akbarzadehlaleh et al.,
2016). The resulting suspension was then centrifuged for 5mins and the process repeated.
Quantitative calibration method could also be used(5).
The buffer for both the DEAE cellulose resin and CM resins were prepared separately
(Padashi et al., 2016). For the DEASE cellulose tris buffer with 20 molar concentration in pH of

ION CHROMATOGRAPHY 3
7.6 and chloride ions as the counter ion was used as the buffer instead of the bis-tris buffer or
bis-tris propane buffer with 20 molar concentration and chloride ions as the counterions (Padashi
et al., 2016). Formic acid buffer with 50 molar concentration in pH of 3.6 and sodium ions as
the, on the other hand, was used as the buffer for the CM-cellulose resin instead of the lactic
acid(Padashi et al., 2016). For the counterion concentration determination for the DEAE
cellulose resin, five different concentration from its concentration range of 0.05 to 0.25 of the tris
buffer was taken (Wu, Li, Shi, Zhang, & Yao, 2016). 0.5ml of each of the 5 buffers then added to
equal amount of the DEAE cellulose resin followed by 0.5ml of the plasma and the final mixture
shaken to create suspension which is then centrifuged for 5minutes to remove the suspension.
The results obtained showed the improved method of albumin purification using sodium
carboxymethyl cellulose resin (stationary phase) which is better than the Diethylaminoethyl
cellulose resin (Wu, Li, Shi, Zhang, & Yao, 2016). The results also showed that DEAE and CM-
cellulose as the two stationary phase used in the protein separation. Diethylaminoethyl cellulose
being an anion exchanger while carboxymethyl cellulose is a cation exchanger (Raoufinia,
Balkani, Keyhanvar, Mahdavipor, & Abdolalizadeh, 2018). Ion exchange chromatography takes
place as a result of the electrostatic attraction between the buffer dissolved proteins and the
oppositely charged cellulose resin, usually composed of spherical inert beads with charged
functional groups which are then neutralized by the counterions.
The size of the pores of the two different types of resins and also the isoelectric point of
HAS could the most probable explanation why CM gave a higher percentage of albumin from
the protein compared to the 75% of the albumin obtained from the DEAE cellulose exchange
resin (Raoufinia, Balkani, Keyhanvar, Mahdavipor, & Abdolalizadeh, 2018). The pore size of
the CM resin ranging (250-350) micrometer while that for DEAE cellulose resin ranging (60-