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C8102 Special methods - laboratory course
Comparison of capillary and conventional HPLC
1 Introduction
High performance liquid chromatography (HPLC) belongs to one of the most powerful analytical
techniques. At first, sample is introduced on the cylindrical column filled with porous material (stationary
phase) in the flow of solvents (mobile phase). Interaction differences in between sample and mobile and
stationary phase than allow separation of individual compounds and their subsequent elution from the
column.
The HPLC instrument (Figure 1) consists from reservoir of mobile phase, high pressure pump, sample
injector, HPLC column, detector, and computer with a software allowing processing of individual peaks.
Figure 1. Basic scheme of HPLC instrumentation
Proper selection of stationary and mobile phase is crucial in development of HPLC method and controls
dominant retention mechanism. In reversed-phase mechanism the stationary phase is nonpolar (modified by
C4, C8, or C18 alkyl chains) with polar mobile phase (combination of acetonitrile, methanol,
tetrahydrofuran, and water). On the other hand, normal phase liquid chromatography takes place on polar
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stationary phase (silicagel, aluminum oxide) with nonpolar mobile phase (hydrocarbons). The combination
of both is hydrophilic interaction liquid chromatography (HILIC) with polar stationary phase and polar
mobile phase. Mobile phase in HILIC is highly organic with low (2 – 20%) concentration of water. Water
in the mobile phase forms aqueous layer on the surface of the stationary phase where separation takes place.
Hydrophilic interaction chromatography offers retention for highly polar compounds that are not retained
in reversed-phase mechanism [1].
There are two main types of stationary phases in HPLC that differ in internal structure: spherical particles
and monolithic stationary phases [2]. Figure 2A shows main morphology difference in between these two
types of columns. Monolithic stationary phases are formed by one piece of porous material that fills the
whole volume of the cylindrical column. Based on the chemistry, there are two main types of the monolithic
stationary phases: monolithic stationary phases based on organic polymers and inorganic monolithic
stationary phases formed from sintered silica (Figure 2B and 2C). While silica monoliths excel in the
separation of small molecules, the main application area of (commercially available) polymer monoliths is
in the fast gradient separations of large natural and synthetic polymers [3].
Figure 2. (A) Difference in between packed and monolithic HPLC column, (B) internal structure
of polymer-based monolith, (C) internal structure of silica-based monolithic stationary phase.
Dopamine belongs to one of the most important neurotransmitters and undesired changes in its
metabolism result in serious illnesses such as depression, schizophrenia, Parkinson disease, and tumors [4].
Dominant dopamine biosynthesis pathway starts at hydroxylation of tyrosine to form
3,4-dihydroxyphenylalanine which decarboxylation leads to dopamine. Alternatively, dopamine can be
biosynthesized by oxidation of tyramine. The main endproducts of dopamine degradation are
3,4-dihydroxyphenylacetic and homovanillic acids. Dopamine also serves as a precursor of epinephrine and
norepinephrine. Phenolsulfotransferases and uridine diphosphoglucuronosyltransferases catalyze
conjugation reactions with phosphate and glucuronic acid, respectively [5].
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The aim of this laboratory task is determination of dopamine in urine by using both a conventional and
capillary HPLC system. While conventional HPLC system uses commercially available column with silicabased
monolithic stationary phase, in case of modular capillary HPLC system the separation takes place on
home-made polymer-based monolithic capillary column.
2 Experimental part
2.1 Chemicals
Acetonitrile, water, trifluoracetic acid, dopamine ware purchased from Sigma Aldrich (St. Louis, MO,
USA).
2.2 Instrumentation
Conventional HPLC: Shimadzu HPLC system with high pressure pump, injector (Vi = 20 l), column
compartment and detector. Column: 2 x Onyx Monolitic C18, 150 x 4.6 mm.
Capillary HPLC: Modular system with high pressure
pump, flow splitter, injector (Vi = 4 nl), and capillary detector.
Column: Polymethacrylate monolithic capillary column 180
x 0.32 mm.
2.3 HPLC
Teaching assistants will provide all necessary information
about setup and control of the HPLC systems. In general,
prepared samples are introduced in the flow of the mobile
phase by using injector. After elution of the dopamine form
the column, peak height and area are determined using
chromatographic software. Each analysis
is repeated in triplicate.
2.4 Solid-phase extraction
Commercially available SPE cartridges using a pHdependent
ring formation reaction of cis-diol group with
boronic acid functionality attached at the surface of SPE
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material (BondElut PBA, Agilent, Palo Alto, CA, USA) are used to extract dopamine from the sample of
human urine. To extract dopamine follow provided protocol.
2.5 Determination of dopamine
Slope of the regression line and standard deviation of residuals (difference in between fitted and
experimental value) is used to determine limits of detection and quantification, as shows Eq. (1)
and (2):
𝑐 𝐿𝑂𝐷 =
3∙𝜎 𝑟
𝑘
(1)
𝑐 𝐿𝑂𝑄 =
10∙𝜎 𝑟
𝑘
(2)
where r corresponds to the standard deviation of residuals and k is a slope of the regression curve.
Calibration curve is also used to determine extracted dopamine in a human urine. To determine
dopamine concentration in the urine by a standard addition method 50, 100, 150, and 200 l
of 2 mg/ml dopamine standard has been added to 1 ml of urine sample prepared as described in
previous section. Determined peak areas are then plotted against concentration of dopamine (mg/l)
and slope and intercept of regression line were used to determine concentration of dopamine in
urine sample:
𝑐𝑆𝐴 =
𝑞
𝑘
(3)
Where q and k are intercept and slope of regression line, respectively.
To compare concentration of dopamine obtained by conventional and capillary HPLC systems
Lord test is applied:
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𝑢 =
|𝑋 𝐴−𝑋 𝐵|
𝑅 𝐴+𝑅 𝐵
(4)
Where XA and XB are mean values of dopamine concentration determined on individual
instruments, and RA and RB are interval of maximal – minimal values, respectively. The parameter
u is compared with critical tabled value for number of experiments in agreement with following
table. In case u > ucritical, the difference in between two instruments is statistically significant and
methods do not provide the same results.
n 2 3 4 5 6 7 8 9 10
ucritical 1.714 0.636 0.404 0.306 0.250 0.213 0.186 0.167 0.152
3 Tasks
1) Prepare 100 ml of 5% aqueous acetonitrile with 0.1% TFA and 1000 ml of 40% acetonitrile with
0.1% TFA.
2) By subsequent dilution of provided sample of dopamine (2 mg/ml) prepare calibration curve
solutions with concentration of dopamine being 0.125, 0.25, 0.5, 1.0, and 2.0 mg/ml.
3) Extract dopamine from sample of human urine by protocol provided in Experimental section.
4) Plot peak height and area vs. concentration of dopamine and construct calibration curve with linear
fit of experimental data. Use error bars for each point of the curve. Use calibration curve to determine
concentration of dopamine in the urine sample. Present calibration curve as an individual plot.
5) Plot peak height and area vs. concentration of added dopamine and construct curve with linear fit of
experimental data. Use error bars for each point of the curve. Use standard addition method to
determine concentration of dopamine in the urine sample. Present sample addition method as an
individual plot.
6) Compare conventional and capillary HPLC systems in determination of dopamine. Focus on limit
of detection and quantification, precision of dopamine determination, robustness and solvents
consumption by both HPLC systems. Use Lord test to compare both instruments.
7) Critically evaluate obtained results.
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References
[1] Snyder, L. R., Kirkland, J. J., Dolan, J. W., Introduction to Modern Liquid Chromatography, Third
edition, Wiley, Hoboken, New Yersey 2009.
[2] Neue, U. D., HPLC Columns: Theory, Technology, and Practice, Wiley, New York 1997.
[3] Urban, J., J. Sep. Sci. 2016, 39, 51-68.
[4] Marc, D. T., Ailts, J. W., Campeau, D. C. A., Bull, M. J., Olson, K. L., Neuroscience & Biobehavioral
Reviews 2011, 35, 635-644.
[5] Meiser, J., Weindl, D., Hiller, K., Cell Communication and Signaling 2013, 11, 1-18.