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Abstract

High-performance liquid chromatography was used to determine the concentration of sucrose,


glucose. Fructose in honey sample. It was found out that fructose would elute first, followed by
glucose, and then sucrose. Different concentrations of mixed standards of sucrose, glucose, and
fructose were used as standards. It was found out that the honey sample contained 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.

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.

Figure 4.2. Basic components of HPLC

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

B. Preparation of sample standards

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.

C. Reading of Standards and Sample in HPLC Instrument


The standard and samples were read in the HPLC system by obtaining 20µL of the samples.

II. Results and Discussion

High-performance liquid chromatography (HPLC) was used in quantifying the concentration of


different sugars in honey sample. Figure 4.2 shows the basic parts of an HPLC instrument. Solvent
reservoir is important since solvents are necessary for the separation of the components and producing
elutes. Modern LC apparatus has provisions to remove dissolved gases and dust from the liquids which
can lead to irreproducible flow rates and band spreading (Skoog, 2007). The pump system puts pressure
to the mobile phase up to 6000 psi in order to achieve reasonable column elution times. Reproducible
flow rate is a measure on the quality of the pump (Harris, 2007). The typical flow rate is 0.1 to 10
mL/minutes. The injection system introduces small amount of sample (0.1 to 500 µL) into the carrier
stream under high pressure. The chromatographic column is typically 10-30 cm in length containing a
packing of 5-10 µm diameter. The column contains the stationary phase and this is where the separation
of components happen. LC detectors has two kinds: the bulk-property detectors and solute-property
detectors. Bulk-property responds to a mobile phase bulk property while solute-property detectors
respond to some property of solutes. Bulk-property detectors include refractive index, dielectric
constant while solute-property detectors include UV absorbance and fluorescence (Skoog ,2007). The
type of detector that will be used will depend on the sample to be analyzed. Detectors available for
HPLC are UV, IR, refractive index, fluorescence, conductivity, mass spectrometry, and electrochemical.
An ideal detector should be sensitive to low concentrations, provide linear response, and does not
broaden the eluted peaks. For the experiment, refractive index was used as a detector. It has the
advantage of responding to nearly all solutes. The principle behind this is the difference in refractive
index of the two solution that allows the bending of the incident beam. The resulting displacement of
the beam with respect to the photosensitive surface of detectors causes variations in the output signal
(Skoog, 2007).

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.

Standard Peak Retention time Area Height Width at base


line (in time)
Distilled Water 1 2.254 9577 699 0.5365853659
Glucose 1(void) 2.277 3326 568 0.1720430108

2 4.44 2901 102 0.6451612903


Fructose 1 (void) 2.277 9168 657 0.3829787234

2 4.007 2781 186 0.5957446809


Sucrose 1(void) 2.277 5353 604 0.2150537634
2 6.065 3003 148 0.6746987952
Xylose 1 2.264 6125 666 0.2173913043
2(void) 3.442 2609 188 0.5652173913
As we can see from table 4.1, the order of retention time in increasing order is xylose, sucrose,
glucose, and fructose. Retention time is the time it takes after injection for a solute to reach the
detector. We can see that xylose is the least polar having the least –OH bond while sucrose is the most
polar having the most –OH bond. Glucose and fructose have almost the same retention time, but
fructose has a 5-membered ring, which is smaller, that is why it is eluted first. The retention time
produced by distilled water is called the dead time, which represents the elution volume or retention
time for unretained species.

Table 4.2 Data on the capacity factor (k’), and efficiency (N) on glucose, fructose, sucrose, and xylose.

Standard k’ N (using tangent)


Glucose 0.9499341238 757.7907841
Fructose 0.7597716293 723.8334199
Sucrose 1.663592446 1292.890439
Xylose 0.5203180212 593.3496384
Using the data on the retention time of the dead time and retention time of the solutes, we can
calculate the capacity factor (k’) of each solute. It is used in measuring the retention of an analyte on the
chromatographic column. A high k’ value indicates that the sample is highly retained and spent a
significant amount interacting with the stationary phase. The formula used for k’ is:

Equation 4.1

Where: tr = retention time


to = dead time

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

Where: tr = retention time


wb = peak width

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

Where: H = plate height


A = eddy diffusion
B = longitudinal diffusion
C = non-equilibrium mass transfer
µ = flow rate

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

Concentration Peaks Retention time Area Height Width


Distilled water 1 2.257 10501 683
10 mg/mL 1 2.263 5801 629 0.243902439
2 (fructose) 4.008 541 47 0.3902439024
3 (glucose) 4.398 1043 72 0.5365853655
4 (sucrose) 6.018 1377 78 0.6829268293
20 mg/mL 1 2.28 4190 595 0.1720430108
2 4.04 2152 145 0.4086021505
3 4.452 3218 185 0.5161290323
4 6.093 2924 149 0.688172043
30 mg/mL 1 2.261 6763 592 0.2553191489
2 4.045 3468 218 0.4255319149
3 4.48 4817 279 0.5531914894
4 6.158 4417 224 0.7654574468
40 mg/mL 1 2.297 7650 580 0.347826087
2 4.085 4901 298 0.4782608696
3 4.557 6802 386 0.5652173913
4 6.2 6835 311 0.8695652174
50 mg/mL 1 2.267 6538 525 0.3404255319
2 4.085 4261 285 0.6382978723
3 4.552 6138 369 0.5531914894
4 6.258 6114 294 0.8510638298

Table 4.3 Data on capacity factor (k’) of the different concentrations of mixed standards of fructose,
glucose, and sucrose.

Concentration, mg/mL Fructose Glucose Sucrose


10 0.7711003093 0.9434379143 1.659301812
20 0.7719298246 0.9526315789 1.672368421
30 0.789031402 0.9814241486 1.72357364
40 0.7784066173 0.9838920331 1.6991728834
50 0.801940891 1.007940009 1.760476401
Table 4.4 Data on selectivity (α) of the different concentrations of mixed standards of fructose, glucose,
and sucrose.

Concentration, mg/mL α between peaks 2 and 3 α between peaks 3 and 4


10 1.223495702 1.758782201
20 1.234090909 1.755524862
30 1.243834081 1.756196485
40 1.263982103 1.72699115
50 1.256875688 1.746608315
The selectivity or α the peaks of the different concentration of standards was calculated. The α
is the ability of to distinguish between sample components. The formula for calculating the α is:

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.

Concentration, mg/mL Fructose Glucose Sucrose


10 1687.730724 1074.862187 1242.441646
20 1564.160869 1190.457009 1254.262067
30 1445.748529 1049.361268 1035.517033

40 1167.278284 1040.032731 813.3904


50 655.3258672 1083.361778 865.1010387
Table 4.6 Data on the resolution of the different concentrations of mixed standards of fructose, glucose,
and sucrose.

Concentration, mg/mL Resolution between peaks 2 Resolution between peaks 3


and 3 and 4
10 0.8415789482 2.656800002
20 0.8910697674 2.725232143
30 0.8891063274 2.545029123
40 0.9046666666 2.290242424

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.

Table 4.7 Data on the calibration curve for fructose.

Concentration, mg/mL Area


0 0
10 541
20 2152
30 3468
40 4901
50* 4261

Table 4.8 Data on the linear analysis of fructose calibration curve.


Parameter Value
Slope 127.29

Y-intercept -333.4
R2 0.9821606131

Table 4.9 Data on the calibration curve for glucose

Concentration, mg/mL Area


0 0
10 1043
20 3218
30 4817
40 6802
50* 6138

Table 4.10 Data on the linear analysis of glucose calibration curve.

Parameter Value
Slope 173.78

Y-intercept -299.6
R2 0.9908233245

Table 4.11 Data on the calibration curve for sucrose.

Concentration, mg/mL Area


0 0
10 1377
20 2924
30 4417
40 6835
50* 6114
=

Table 4.12 Data on the linear analysis of sucrose calibration curve.

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.

Sample (DF = 10) Area Concentration, mg/mL


Fructose 3598 308.8537984
Glucose 4161 256.6808609
Sucrose 5217 326.0562537

Table 4.14 Data on the k’ of fructose, glucose, and sucrose of the honey sample.

Sample Dead time Retention time K'


Fructose 2.27 4.047 0.78281938233
Glucose 2.27 4.472 0.9700440529
Sucrose 2.27 6.177 1.721145374
Table 4.15 Data on the N 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.

III. Summary and Conclusion

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.

IV. Literature Cited

Chromacademy. (n.a). The Theory of HPLC: Chromatographic Parameters. Crawford Scientific.


Retrieved at
https://www.chromacademy.com/lms/sco2/Theory_Of_HPLC_Chromatographic_Parameters.pdf

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