Comparative Analysis of Cell Culture Separation Methods

This report provides an overview of various separation methods used in cell culture, with a specific focus on High-Performance Liquid Chromatography (HPLC) and Hydrophilic Interaction Liquid Chromatography (HILIC). It compares the two methods, highlighting their differences and applications for different materials. The report discusses the principles of reverse-phase HPLC, the separation of beta-lactam antibiotics, and the use of HPLC for separating tetracycline and fluoroquinolones. It also details the HILIC method, including stationary phases, interactions between stationary and mobile phases, and its application in separating glycosaminoglycans like enoxaparin. The report further analyzes the impact of pH on the mobile phase and the retention of analytes in HILIC separations, concluding with a discussion on the resolution of the dp2 molecule and the influence of stationary phases. The report references several scientific publications to support its findings and analyses.

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Cell Culture Techniques
Name
Date of Submission

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1. The purpose of the experiments performed in week 6 was to understand various
separation methods used in various analytes. In specific, chromatographic methods, more so the
HPLC and HILIC. These two separation methods were compared to find the differences and
determine which method is better for certain materials and not the other. In this case, it was
noted that the HILIC makes use of hydrophilic stationary phases and a reverse phase eluent. The
most common mobile phase used in HILIC is acetonitrile alongside some small amounts of water
1. The principles of reverse phase HPLC is that it uses a gradient elution instead of the isocratic
elution. The biomolecules adsorb to the surface of a reverse phase matrix strongly under the
aqueous environment. Additionally, the biomolecules desorb from the matrix at a narrow
window. The beta lactam antibiotics can be separated using HPLC because they are made up of
various components of varied ionic strengths. These can easily be carried by the mobile phase
and eluted accordingly.
2. Tetracycline is an antibiotic which can be easily separated using the RPHPLC and not
HILIC. This molecule is a broad spectrum antibiotic which acts against a number of pathogenic
organisms. Tetracycline is an adaptive compound which can modify itself with relative ease via
tautomerism in response to various environmental conditions 2. These environments could have
effects to the coordination behaviors of tetracycline for instance with various metal ions. This is
because tetracycline has several potential metal binding sites and hence the ease of separation.
Thus, the separation methods with tetracycline shows high limits of detection, high linear ranges
and low time of analysis since it does not require an extraction phase. Fluoroquinolones: a
mixture of fluoroquinolones such as ciprofloxacin and sparfloxacin can be separated using the
HPLC. In this case, the mobile phase is composed of acetonitrile and trifluoroacetic acid 3.

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3. The HILIC needs a hydrophilic type of a stationary phase in order to adsorb water
layers and allow the analytes process of partitioning. The hydrophilic nature of the material used
to pack the column determines the thickness of the water layer and hence the rate of retention the
analyte. Additionally, the interactions between the stationary phase and the analyte include
hydrogen bond formation, and electrostatic forces 4. This therefore highlights the need for
considering the chemistry of the stationary phase selected for the process. the current HILIC
stationary phases used are bare silica and other polar chemically bonded phases that have
ionizable groups linked to the surface of silica. The ligands used in this separation process can
undergo electrostatic interactions and also add extra dimensions to the separations during the
separation of ionizable materials. The HILIC stationary phases are: neutral- there have a polar
surface which does not contain any electrostatic forces, the second is the charged phase-has
strong electrostatic forces since it has either anionic or cationic groups. Third, is the zwitterion
stationary phase which has weak electrostatic interactions due to the presence of both anionic as
well as cationic groups.
4. The glycosaminoglycan need to have their components analyzed in order to determine
their structures. Enoxaparin is an example or type of glycosaminoglycan which is used clinically
as lovenox as an anticoagulant medication. Thus enoxaparin primarily treats deep vein
thrombosis as well as pulmonary embolism. Therefore, HILIC has been widely used in the
separation of carbohydrates including glycosaminoglycan. In this method, the polar materials are
separated on the basis of the stationary phases by use of water miscible organic solvents, while
water itself is an elutropic solvent. The enoxaparin is a low molecular weight carbohydrate in
which the original reducing end and non-reducing end is identified by reducing the reducing
portion followed by its hydrolysis by use of hydrogen peroxide. This compound is neither a

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single chemical nor a definite mixture of a compound 5. Thus it presents a feature of
heterogeneity and high level of variations which depends on the source and origin. It is also
common that individual polysaccharide chains could have similar physical and chemical
properties but it is not possible to separate them using the current separation methods. This is the
common problem which is encountered when separating enoxaparin by the use of HILIC
separation method. This close proximity in the chemical and physical composition of the
components of this drug makes it difficult to separate the analystes using the mobile and
stationary phases.
5. the HILIC separation method offers a way of separating the small and polar molecules
using polar stationary phases. The dp2 molecule had a high resolution because the it was polar
and thus had a high solubility in the aqueous mobile phase that is applied in HILIC. This
therefore makes the dp2 overcome the problem of low material and analyte solubility which are
common in other separation methods like HPLC. Moreover, the HILIC method can be applied
for both amphiphilic, and the uncharged highly hydrophilic molecules. These molecules are said
to be too polar such that they cannot be retained in a RPLC. Such molecules however contain
enough charges which allows for the electrostatic interactions during HILIC separations 6. The
variations in the obtained resolutions of dp2 in HILIC and RPHPLC is could be due to the nature
of the stationary phases used. For instance, the HILIC had a high precision and used a bare silica
gel as the stationary phase, which is the most recommended form of stationary phase. However,
the RPHPLC had a low precision probably because the stationary phase used an unbounded
silica. In HILIC, the peak reading was at the seventh minute while in the RPHPLC a lower peak
was observed at the fifth minute.

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6. the pH of the mobile phase plays significant roles in the retention functions of HILIC
separation process. this is because pH is capable of influencing the electrical charging nature of
the stationary phase and the ionizable solutes 5. This in turn can affect the thickness of the
stagnant and enriched aqueous layer on the stationary phase surface. Additionally, pH can also
cause additional ionic interactions during the material retention. An experiment was conducted to
determine the effect of pH on the mobile phase, on the retention of the analytes. In this case, the
pH of the mobile phase was changed between 5.8 to 6.8 but all other components of the mobile
phase remained constant. Upon the addition of buffer to acetonitrile, the pH was adjusted and the
findings were that there was a decrease in the retention time of glimepiride as the pH levels
increased. During the pH changes from 5.8 to 6.8, the stationary phase had positively charged
amino acids, and hence could cause some form of electrostatic attractions with the negatively
charged solutes in the mobile phased and hence affect the retention abilities. This is an indication
of a possible mixed mode of hydrophilic interactions between the stationary phase, mobile phase
and the solutes. This therefore supports the statement that it is better if the pH of the mobile
phase is left unadjusted during a HILIC experiment.

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References
1. Logotheti M, Theochari K, Kostakis M, Pasias IN, Thomaidis NS. Development and
validation of a HILIC-UV method for the determination of nucleotides in fish samples.
Food chemistry. 2018 May 15;248:70-7.
2. Abdulghani AJ, Jasim HH, Hassan AS. Determination of tetracycline in pharmaceutical
preparation by molecular and atomic absorption spectrophotometry and high performance
liquid chromatography via complex formation with Au (III) and Hg (II) ions in solutions.
International journal of analytical chemistry. 2013;2013.
3. Cavazos-Rocha N, Carmona-Alvarado I, Vera-Cabrera L, Waksman-de-Torres N,
Salazar-Cavazos MD. HPLC method for the simultaneous analysis of fluoroquinolones
and oxazolidinones in plasma. Journal of chromatographic science. 2014 Mar
3;52(10):1281-7.
4. Richardson H, Bidlingmeyer BA. Bare silica as a reverse‐phase stationary phase: Liquid
chromatographic separation of antihistamines with buffered aqueous organic mobile
phases. Journal of pharmaceutical sciences. 1984 Oct 1;73(10):1480-2.
5. Yates EA, Rudd TR. Recent innovations in the structural analysis of heparin.
International journal of cardiology. 2016 Jun 1;212:S5-9.

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6. Zhou S, Zuo P, Zuo Y, Deng Y. A rapid hydrophilic interaction liquid chromatographic
determination of glimepiride in pharmaceutical formulations. Saudi Pharmaceutical
Journal. 2017 Sep 1;25(6):852-6.