Evaluation of Ion Chromatography Techniques for Albumin Purification

This report details an experiment on albumin purification using ion chromatography. The study compares two methods: carboxymethyl cellulose (CM) and diethylaminoethyl cellulose (DEAE) resin exchange. The CM-cellulose resin method proved more effective, yielding a higher percentage of purified albumin. The research utilized a conductivity detector and qualitative calibration. The report also discusses the buffers used, including Tris buffer for DEAE cellulose and formic acid buffer for CM-cellulose. The results showed that CM-cellulose resin is better than DEAE cellulose resin for albumin purification. The report also describes the role of various instruments like centrifuge, shaker, SDS-page, pH meter, conductivity detector, stopwatch and voltmeter in the research. The study highlights the importance of buffer selection in the process, with formic acid and tris buffer being the most suitable for CM-cellulose and DEAE cellulose resins respectively. The report concludes that the CM-cellulose method is more beneficial in albumin purification than the DEAE cellulose method.

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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

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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-

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130) micrometer (Raoufinia, Balkani, Keyhanvar, Mahdavipor, & Abdolalizadeh, 2018). The
pH of the HAS was found to be 4.8 while that for CM-cellulose resin was 3.8. Other impurities
were then deleted off within this pH range resulting in a pure HSA (Human Serum Albumin)
being produced to achieve quality control control requirements(8) (Raoufinia, Balkani,
Keyhanvar, Mahdavipor, & Abdolalizadeh, 2018). Both of the two resins DEAE and CM-
cellulose resins are signed with the CAS number for quality control purposes.
TABLE 1: the instrument and the role in the research.(4)
INSTRUMENT ROLE IN THE RESEARCH
Centrifuge For the removal of the suspensions
Shaker For creating suspensions in the sample
SDS-page For sample analysis
pH meter Checking pH changes for the buffer
solution
Conductivity detector For detecting the analyte in the sample
Stopwatch For recording the time of the reaction
Voltmeter For current measurements
Finally, the choice for the buffer for this research made the whole process very unique
(9) (Raoufinia, Mota, Keyhanvar, Safari, Shamekhi, &Abdolalizadeh, 2016). For the DEAE
cellulose resin which uses bis-tris buffer or bis-tris propane buffer with 20 molar concentration
and chloride ions as its counter –ion as the best buffer, the research however used tris buffer with
20 molar concentration with a pH of 7.6 and chloride ions as the counterion (Raoufinia, Mota,
Keyhanvar, Safari, Shamekhi, &Abdolalizadeh, 2016). For the CM-cellulose resin, the research

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chose on formic acid as the buffer with 50 molar concentration in a pH of 3.6 and the sodium
ions as the counter –ions instead of the lactic acid which is considered the best making the whole
research more unique(9). The study illustrated that albumin purification by the use of CM-
cellulose resin is much more beneficial compared to the DEAE cellulose resin.

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References
Raoufinia, R., Mota, A., Keyhanvar, N., Safari, F., Shamekhi, S. and Abdolalizadeh, J., 2016.
Overview of albumin and its purification methods. Advanced pharmaceutical bulletin,
6(4), p.495.
Raoufinia, R., Balkani, S., Keyhanvar, N., Mahdavipor, B. and Abdolalizadeh, J., 2018. Human
albumin purification: a modified and concise method. Journal of Immunoassay and
Immunochemistry, 39(6), pp.687-695.
POLTEP, K., TESENA, P., YINGCHUTRAKUL, Y., TAYLOR, J. and WONGTAWAN, T.,
2018. Optimization of a serum albumin removal protocol for use in a proteomic study to
identify the protein
Wu, Q.C., Lin, D.Q., Shi, W., Zhang, Q.L. and Yao, S.J., 2016. A mixed-mode resin with
tryptamine ligand for human serum albumin separation. Journal of Chromatography A,
1431, pp.145-153.
Akbarzadehlaleh, P., Mirzaei, M., Mashahdi-Keshtiban, M., Shamsasenjan, K. and Heydari, H.,
2016. PEGylated human serum albumin: a review of PEGylation, purification and
characterization methods. Advanced pharmaceutical bulletin, 6(3), p.309.
Padashi N, Arjmand M, Rajaei S, Dabbagh, A (2016). Purification of Human Serum Albumin by
Ion Exchange Chromatography. J Cell Mol Anesth, 1(4):158-62.