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I. Introduction
Chromatography is an important method especially when want to separate, identify, and determine
closely related components in a mixture. In a chromatographic separation, the sample is dissolved in a
mobile phase, which can be a gas, a liquid, or a supercritical fluid. The mobile phase is forces through an
immiscible stationary phase, which is fixed in place in a column or on a solid surface (Skoog, 2007). The
components of the sample are distributed between the mobile phase and the stationary phase to
varying degrees. Column chromatography is a classification of chromatography based on the physical
means by which the stationary and mobile phases are brought into contact. The stationary phase in
column chromatography is held in a narrow tube through which the mobile phase is forced under
pressure.
There are several types of chromatography based on the mechanism of interaction of the solute with
the stationary phase. In adsorption chromatography, solute is adsorbed on the surface of the solid
particles. In partition chromatography, the solute equilibrates between the stationary liquid and the
mobile phase, which is a flowing gas in gas chromatography. In ion-exchange chromatography,
electrostatic force occurs and the solute ions in the opposite charge are attracted to the stationary
phase. In molecular exclusion chromatograph, which is also called gel filtration or gel permeation
chromatography, separates molecule by size. Lastly, in affinity chromatography, it employs specific
interaction between two different kinds of molecules that are covalently attached to the stationary
phase (Harris, 2007). Result of chromatography is called a chromatogram, which shows the detector
response as a function of elution time.
Liquid chromatography is important in separating components that are not volatile enough to be
separated by gas chromatography. Types of liquid chromatography depends on the molecular weight of
solute, solubility of solute, polarity of solute, and its ionic/non-ionic character. High-performance liquid
chromatography (HPLC) is a kind of liquid chromatography in which the solvent is force through the
column containing very fine particles (stationary phase) using a high pressure up to 3000 psi to give
high-resolution separations.
Figure 4.1 Selection of LC Modes
The basic components of HPLC are: solvent reservoir and pump system, injection port, column,
detector system and a readout.
I. Methodology
A. Sample Preparation
A stock solution of the sample was made by diluting 2.5 mL honey in a 25-mL volumetric flask.
From the stock solution, sample solutions with DF 10, 50, 100, 200, and 250 were made by serial dilution
Standard solutions of glucose, fructose, sucrose, and xylose were prepared. The concentration
of the standards is 25 mg/mL
A stock solution containing 50 mg/mL each of glucose, sucrose, and fructose was made by
weighing 5 g each of the sugars and diluting it to mark in a 100-mL volumetric flask. From the stock
solution, different concentrations (0, 10, 20, 30, 40, and 50 mg/L) of the three sugars was made and
mixed to obtain a solution containing the three sugars with the same concentration.
The sample was analyzed for its glucose, fructose, and sucrose concentration. In analyzing using
HPLC, it is important to know the type HPLC used if it is either normal phase or reversed phase. Because
the type of components that would elute first depend if it is either normal phase or reversed phase. In
normal phase, a polar stationary phase is used and a non-polar mobile phase is used. In reversed-phase,
that stationary phase is non-polar while the mobile phase is polar. That is why in normal phase, the least
polar component is eluted first so increasing the polarity of mobile phase would cause a decrease in
elution time. On the other hand, the most polar component elutes first in reversed-phase. Increasing the
mobile-phase polarity increases the elution time (Skoog, 2007).
Figure 4.3 Structure of sucrose, fructose, glucose, and xylose (from left to right).
Table 4.1 Data on the results of chromatography of distilled water, glucose, fructose, sucrose and xylose.
Table 4.2 Data on the capacity factor (k’), and efficiency (N) on glucose, fructose, sucrose, and xylose.
Equation 4.1
The recommended value of k’ is between 1 to 5. A value less than 1 is unreliable since it is too rapid for
accurate determination, or analytes may be eluting with other components or solvent. A value greater
than 5 provides minimal resolution (Chromacademy, n.a.).
A measure of efficiency of the column is in terms of number of plates or N. the number of
interactions that a solute has during its residence in the column. Meaning, the higher the N, the more
efficient the column. The equation for calculating N is:
Equation 4.2
We can calculate the plate height (HETP) when we determined the N. The plate height can be
calculated using the equation N= L/H. Meaning, the higher the number of plates, the lower the plate
height. The smaller the plate height, the narrower the bandwidth. The ability of a column to separate
components of a mixture is improved by decreasing plate height. The plate height is directly related to
the variance of chromatographic band. The van Deemter equation relates the column and flow rate with
plate height.
H = A + B/µ + Cµ
Equation 4.3
Eddy diffusion arises from the different microscopic flow streams that the solvent follows between
particles within the columns. Band broadening is caused by multiple flow paths through a path column.
The solvent moves faster in wide paths than narrow paths. Longitudinal diffusion is band broadening
due to the diffusion of solute along the length of a column in the flowing mobile phase. Non=equilibrium
mass transfer refers to differing flow rates for different parts of a single flow stream of path between
surrounding particles.
The concentration of fructose, glucose, and sucrose in honey sample was calculated. A different
concentration of mixed standard containing fructose, glucose, and sucrose was made. The retention
time, area, and height were determined for each eluted peak. The width of the peaks were calculated by
measuring the distance in the chromatogram and using ratio and proportion.
Table 4.3 Data on the results of chromatography of different concentrations of mixed standards of
glucose, sucrose, and fructose
Table 4.3 Data on capacity factor (k’) of the different concentrations of mixed standards of fructose,
glucose, and sucrose.
Equation 4.4
The higher the α, the better the separation and the better the separation between the apex of each
peak. The selectivity should always be greater than 1. When α = 1, it means the two peaks are co-
eluting.
Table 4.5 Data on the plate efficiency of the different concentrations of mixed standards of fructose,
glucose, and sucrose.
50 0.7838928571 2.467138461
The column resolution tells us how far apart two bands are relative to their widths. It is a
measurement of the ability of the column to separate two analytes. There are three parameters that
affect the resolution of a column. The capacity factor (k’) of 1-5 is ideal in order to achieve a good
resolution. The selectivity or α, which describes the relative retention of the components by the
stationary phase, can also affect the resolution. Lastly, the efficiency (N), which relates the band
broadening and retention volume, can affect the resolution.
Equation 4.5
To quantify the amount of sucrose, fructose, and glucose of the sample, the concentration vs.
area of each sugar was plotted and a calibration curve was made. A point was taken for fructose,
glucose, and sucrose (50 mg/mL) to obtain a more linear calibration curve.
Y-intercept -333.4
R2 0.9821606131
Parameter Value
Slope 173.78
Y-intercept -299.6
R2 0.9908233245
Parameter Value
Slope 167.1
Y-intercept -231.4
R2 0.9868718076
Table 4.13 Data on the concentration of fructose, glucose, and sucrose of honey sample.
Table 4.14 Data on the k’ of fructose, glucose, and sucrose of the honey sample.
Sample Width N
Fructose 0.45833333 1247.451044
Glucose 0.625 819.1501926
Sucrose 0.7916666667 974.0706705
After interpolating the areas obtained from the sample to the calibration curve, the
concentration of fructose, glucose, and sucrose obtained are 308.8537984 mg/mL, 256.6808609 mg/mL,
and 326.0562537 mg/mL, respectively. The efficiency of fructose, glucose, and sucrose of the sample are
1247.45, 819.15, and 974.07, respectively.
HPLC method was used to determine the sugar components and to quantify its concentration in
honey sample. It was found out that fructose is first to elute in a normal-phase chromatography,
followed by glucose, and lastly by sucrose. The least polar component is the first to elute in a normal
phase chromatography. The k’ of each component was also calculated and was found that only sucrose
has a k’ that is greater than 1. The recommended value for k’ is between 1 to 5. A value less than 1 is
unreliable since it is too rapid for accurate determination, or analytes may be eluting with other
components or solvent.
Different concentrations of mixed standards containing glucose, fructose, and sucrose were
prepared. After interpolating the data obtained from the sample of honey, it was found out that the
honey sample has a concentration of 308.8537984 mg/mL of fructose, 256.6808609 mg/mL of glucose,
and 326.0562537 mg/ mL of sucrose. The efficiency or N of each component was also calculated and
was found to be 1247.451044 for fructose, 819.1501926 for glucose, and 974.0706705 for sucrose.
Harris, D. (2007). Quantitative Chemical Analysis (7th edition). W.H. Freeman and Company. New
York.
Skoog, D.A., Holler, F. J., Crouch, S. R. (2007). Principles of Instrumental Analysis (6th edition).
Thomson Brooks/Cole. USA