Experiment. HPLC: Separation and Quantification of Components in Diet Soft Drinks
Modified 12/2014
Experiment HPLC: Determination of Vitamin C by Chromatography Objective: The purpose of this experiment is to quantify the amount of vitamin C in an unknown by performance liquid chromatography (HPLC). In order to quantify the vitamin C, a calibration curve must first be established with known concentrations of Vitamin-C. In this experiment, you will determine a set of HPLC conditions suitable for the chromatogram of vitamin C. At the end of this experiment be able to determine the amount of vitamin-C in your unknown by HPLC and understand how to vary experimental parameters to optimize a separation. Equipment Varian HPLC Hamilton Syringe 250 mL Vol flask
Chemicals Diet soda sample (Degas for 20 min) Various vitamin-C standards HPLC-grade methanol 20mM phosphate buffer, pH 3
Safety and Waste Disposal An apron/lab coats and goggles should be worn in the laboratory at all times. The chemicals used in this experiment should pose no significant safety hazards. Good laboratory procedure should be followed at all times.
Discussion The fundamentals of chromatographic separations and a detailed discussion of the application of HPLC is covered in the appropriate chapter in your text. The following discussion summarizes important concepts from these chapters, but the student is encouraged to read the full text. Introduction to HPLC and Instrument Components. High performance liquid chromatography (HPLC) is an important analytical tool for separating and quantifying components in complex liquid mixtures. By choosing the appropriate equipment (i.e. column and detector), this method is applicable to samples with components ranging from small organic and inorganic molecules and ions to polymers and proteins with high molecular weights. The various types of HPLC and their characteristics are summarized in the table below. In this experiment, we will use reversed-phase partition chromatography. Table 1. Various Types and Applications of HPLC TYPE
Partition (reversedphase) Partition (normalphase) Ion Exchange
SAMPLE POLARITY non-polar to somewhat polar non-polar to somewhat polar somewhat polar to highly polar highly polar to ionic
SizeExclusion
non-polar to ionic
Adsorption
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MOLECULAR WEIGHT RANGE 100 - 104
STATIONARY PHASE
MOBILE PHASE
silica or alumina
non-polar to polar
100 - 104
non-polar liquid adsorbed or chemically bonded to the packing material
relatively polar
100 - 104
highly polar liquid adsorbed or chemically bonded to the packing material
relatively non-polar
100 - 104
ion-exchange resins made of insoluble, highmolecular weight solids functionalized typically with sulfonic acid (cationic exchange) or amine (anionic exchange) groups
103 – 106
small, porous, silica or polymeric particles
aqueous buffers with added organic solvents to moderate solvent strength polar to non-polar
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HPLC of Vitamin C
Experiment. HPLC: Separation and Quantification of Components in Diet Soft Drinks
Modified 12/2014
Figure 1. Shows the components of our Varian ProStar 210 HPLC. The system consists of: • reservoirs to hold the solvents used to make up the mobile phases • a programmable quaternary pump that mixes the solvents in the prescribed ratios and pumps them through the column and past the detector • a column compartment that houses the thermostats and the HPLC column. • a diode array detector that monitors the entire UV-Vis spectrum of the column effluent at regular intervals Control of the above components and data acquisition and analysis are performed on a Dell personal computer.
Optimization of Resolution and Column Performance The goal of any HPLC experiment is to achieve the desired separation in the shortest possible time. Time is critical because “time is money” and because as we will see, the more time the sample spends on the column, the more the bands containing the components spread, resulting in reduced resolution. Optimization of the experiment usually involves manipulation of column and mobile phase parameters to alter the relative migration rates of the components in the mixture and to reduce zone broadening. These can generally be optimized fairly independently.
updated 12.04.12
2
HPLC of Vitamin C
Experiment. HPLC: Separation and Quantification of Components in Diet Soft Drinks
Modified 12/2014
Migration Rates The length of time it takes for a given component/solute to travel through the column and be detected is determined by the flow rate of the mobile phase, m, and the partitioning of the solute between the mobile and stationary phases. Since the solute molecules can only travel when they are dissolved in the mobile phase, the greater their concentration in the mobile phase, the faster they will elute. The partition coefficient, K, is defined in equation 1
K=
CS
(1)
CM
where CS is the concentration of the solute dissolved in or adsorbed to the stationary phase, and CM is the concentration of the solute in the mobile phase.
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The quantities CS and CM, however, are rarely determined in chromatographic experiments. Instead, a quantity called the retention factor, k’, is determined. The retention factor for a component A is defined as
k `A =
tR − tM
(2)
tM
where tR is the retention time of component A and tM is the retention time of an un-retained species (uracil), which is also called the dead time. The average rate of linear migration of component A is related to both the flow rate of the mobile phase and the retention factor. €
v = m⋅
1
(1 + k ) ` A
The retention factors should normally lie in a range of 2-5, but for complex mixtures a larger range may be required to separate all the components. The value of the retention factor for a given component depends on the chemical identity of the component and the following experimental variables: € • mobile phase flow rate
• mobile phase composition
• column temperature
• column composition
Zone Broadening: The extent to which the component bands spread as they travel down the column affects the efficiency of the separation. The theoretical plate height, H, is defined in equation 4 and is based on a Gaussian analysis of the peak width, σ, as it exits the column at point L.
H=
where
σ=
σ2
(4)
L
LW 4t R
(5)
€
and W is the width of the peak at the base. The data analysis program on our HPLC actually reports the width at half maximum, W1/2, for each peak rather than the width at the base. Assuming a Gaussian peak shape,
€
(
)
W = 1.6994 W1 /2
so,
€
2
( ) H= 5.540(t ) L W1 /2
(6)
2
(7)
R
updated 12.04.12
3
€
HPLC of Vitamin C
Experiment. HPLC: Separation and Quantification of Components in Diet Soft Drinks
Modified 12/2014
The number of theoretical plates in the column, N, is
N =
L
(8)
H
Efficient columns have small H and large N for a given component. The theoretical plate height is affected by the following experimental parameters:
€
•
mobile phase flow rate
•
diffusion coefficient of the solute in the mobile phase
•
diffusion coefficient in the stationary phase (depends on temperature and viscosity)
•
retention factor
•
diameter of the particles packing the column
•
thickness of the liquid coating on the stationary phase
Resolution The resolution of two adjacent peaks, RS, is determined by their separation and their widths.
RS =
[
2 (tR )B − (tR )A
WA + WB
]=
[ ] (1.6994) #$%(W ) + (W ) &'( 2 (tR )B − (tR )A 1 /2
A
1 /2
(9)
B
In other words, RS depends on both migration rates and zone broadening. A resolution of 1.5 means that the overlap of the peaks is about 0.3%, so conditions should be optimized to achieve at least this resolution if possible.
€
In this experiment, you will adjust only the composition of the mobile phase to optimize the retention factors and resolution. We will not attempt to optimize the zone broadening independently by changing the column or the flow rate.
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HPLC of Vitamin C
Experiment. HPLC: Separation and Quantification of Components in Diet Soft Drinks
Modified 12/2014
The importance of vitamin C in human health is well understood, particularly as an antioxidant and in collagen synthesis. The recommended daily allowance (RDA) for vitamin C is determined as 60 mg/day, sufficient to prevent scurvy and maintain a stable body pool of 1500 mg, much of which comes from fruits and vegetables. Vitamin C action is supplied by L-ascorbic acid and its oxidized form, dehydroascorbic acid, each with equivalent molar activity, with total vitamin C defined as the sum of both forms.
Vitamin C is maintained in its reduced form with dithiothreitol, which also converts endogenous dehydroascorbic acid to ascorbic acid. Total vitamin C is determined as ascorbic acid with LC–UV. HPLC analysis will be performed using a Varian Prostar 210 HPLC equipped with a Varian Prostar 320 detector. Separations will be carried out using an Agilent Eclipse 5 micron C18 4.6 x 150mm column with a phosphate buffered mobile phase (pH 2.5), flow rate of 1 mL/min and detection at 254 nm
Procedure
Notes on Use of HPLC: Your instructor will show you how to use the equipment and the software. At the beginning of the day, first open the valve with the black knob (turn counterclockwise) on the front of the pump manifold. This sends the mobile phase to waste instead of the column. Allow the pump to run for about 10 minutes at 5 mL/min to purge the lines of any air bubbles.
At the end of the day, run an 80% water/20% methanol mixture through the column at 1 mL/min for 10 minutes followed by 100% methanol for 10 minutes. This should flush the column of any potential salt forming materials and stores the column in a compatible solvent.
Procedure
1.
Mobile phase—KH2PO4 (0.5%, w/v), pH 2.5, with dithiothreitol (0.1%, w/v): Weigh 5.0 g KH2PO4 in 1 L volumetric flask and add ca 950 mL HPLC grade water. Add 1.0 g dithiothreitol, stir until dissolved, and adjust pH to 2.5 with concentrated phosphoric acid. Dilute to 1 L with HPLC grade water, and filter through 0.45 um membrane. (This solvent has been prepared for you)
2.
Preparation of Vitamin C standards (10–50 ppm).—Dissolve 5 mg ascorbic acid in a 100 mL water to give 50 ppm. Dilute 2, 4, 6, and 8 mL to 10 mL with water to give 10–40 ppm, respectively. Add 10 mg dithiothreitol to each standard. Prepare fresh each day.
3.
Preparation of unknown ascorbic acid solution. - Weigh around 50mg of your unknown vitamin C (from lab #3) to the nearest 0.1mg, place in a 50ml volumetric flask and then fill to the mark with water. Dilute 5.0ml this unknown sample to 100ml with water. Pipet 2.0ml this solution into small vial and add 2.0mg dithiothreitol to this vial.
updated 12.04.12
5
HPLC of Vitamin C
Experiment. HPLC: Separation and Quantification of Components in Diet Soft Drinks
Modified 12/2014
Operation of the instrument Turn on the HPLC. Turn on the UV detector and let it warm up for about 15minutes. Set the detector range to 1.000 and the wavelength to 254nm.
To Prime the pumps (A and B) - At beginning of the day, first open the valve with black knob (turn the knob the left) on the front of pump manifold. This sends the mobile phase to waste instead of the column. Push arrow to set flow rate 1ml/minutes and time 10 minutes. Press run to purge the line of any air bubbles.
To run the sample -Turn the knob to right. Click File on System control and click the new sample list. Type sample name into the sample list, click data file to save in folder; click begins to choose a method. Clean syringe by rinsing several times with water and with your samples. Draw up sample past the 10uL, and expel down to 10uL; make sure that there is no air in the syringe. Click RUN and switch manual injector to the LOAD position and push 10uL sample through the sample loop; with the syringe still in the injector, turn to the INJECTposition, at which time the instrument will automatically start data acquisition. Leave the injector in the INJECT position, and remove the syringe. Clean syringe the grade HPLC water.
To shut down HPLC- At the end of the day, run 80% water, 20% methanol mixture through the column at 1ml/minutes following by 100% methanol for 10 minutes. This should flush the column of any potential salt
forming materials
and stores the column in a compatible solvent. Calculation 1.
Using the peak areas to calibrate curves from known standards, and then determine the percent of ascorbic acid in your sample.
2.
Determine the error (standard deviation) in your determination of the ascorbic acid percent.
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HPLC of Vitamin C