Chem 137.

1 2L | UPLB 1
Exercise No 4 | 2018

Exercise No 4
Liquid Chromatography

Del Rosario, Heidi Rose B.*, Kintanilla, Luis, Lanceras, Johanssen & Garzon, Irish
*hbdelrosario@up.edu.ph

Abstract
High Performance Liquid Chromatography was used in determining the quantity of fructose, sucrose and
glucose in honey sample. It was found out that the honey sample contains 307.8285653 mg/mL,
260.2776012 mg/mL and 324.3689483 mg/mL fructose, glucose and sucrose respectively. The column
used was evaluated in terms of its efficiency as number of theoretical plates, capacity factor, retention
factor and its resolution. The number of theoretical plates of the reading of the standard sugar solutions
ranges from 600 to 1200. For capacity factor, peaks for fructose and glucose had values less than one
(poor separation) except for the 50 mg/mL peak for glucose and capacity factors for peaks of sucrose had
values greater than 1 thus better separation. For selectivity both separations of fructose with glucose
and glucose with sucrose had values lower than 2, thus not reaching the optimal values for selectivity.
For resolution, there is higher resolution in separation of glucose and sucrose than separation of fructose
and glucose.

I. Introduction
Chromatography is a widely used method for the separation, identification, and determination of the
chemical components in complex mixtures in which the components of a mixture are separated based on differences
in the rates at which they are carried through a fixed or stationary phase by a gaseous or liquid mobile phase. The
stationary phase is a phase that is fixed in place either in a column or on a planar surface while the mobile phase
(may be a gas, liquid or supercritical fluid) is a phase that moves over or through the stationary phase carrying with
it the analyte mixture.
There are two separation modes of chromatography—normal phase and reverse phase. Normal phase
chromatographic separation uses a polar stationary phase with a non-polar or much less polar mobile phase. On the
other hand, reverse phase is the opposite of the normal phase. Reverse phase chromatographic separation uses a non-
polar stationary phase and a polar mobile phase (Waters, 2018).
A recent development in chromatography, early 60’s, was the High-Pressure Liquid Chromatography.
High-performance liquid chromatography (HPLC) is the most versatile and widely used type of elution
chromatography. The technique is used by scientists for separating and determining species in a variety of organic,
inorganic, and biological materials. In this chromatography, the solvent is forced through a chromatographic column
at a pressure up to 3000 psi at room or elevated temperature.
The HPLC system in Figure 4.1 consists of a solvent delivery system, a sample injection valve, a high-
pressure column, a detector, and a computer to control the system and display results. Many systems include an oven
for temperature control of the column (Harris, 2007).

Figure 4.1. HPLC block diagram.

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The exercise aimed the students to be familiar and have experience of an HPLC system. It also aimed to
determine the efficiency of the column and to determine the sugar content of a honey sample.

II. Methodology
A. Sample Preparation
Standard solutions of glucose, fructose, sucrose and xylose were prepared. The concentration of
the standards is 25 mg/mL. Another set of standard solutions containing glucose, fructose and sucrose were
also prepared. The concentration of the sugars in the standards were 10, 20, 30, 40 and 50 mg/mL.
The sample, honey, was prepared in different dilutions.
B. Reading of Standards and Sample in HPLC instrument
Volume of 20 µL of test samples was read in the HPLC system with parameters shown in Table
4.1.

III. Results and Discussion

The basic parts of an HPLC system are shown in Figure 4.1. For the solvent, very pure HPLC-grade
solvents are required to prevent degradation of costly columns with impurities and to minimize detector background
signals from contaminants. A filter is used on the intake tubing in the solvent reservoir to reject micron-size
particles. Sample and solvent are passed through a short, expendable guard column with the same stationary phase
as the analytical column to collect strongly adsorbed species. Organic solvents for normal-phase chromatography
should be 50% saturated with water since normal-phase separations are very sensitive to water in the solvent. For
gradient elution in reversed-phase separations, 10–20 empty column volumes of initial solvent should be passed
through the column after a run to re-equilibrate the stationary phase with solvent for the next run.
For the pumps, the quality of a pump for HPLC is measured by how steady and reproducible a flow it can
produce. A fluctuating flow rate can create detector noise that obscures weak signals. Gradients made from up to
four solvents are constructed by proportioning the liquids through a four-way valve at low pressure and then
pumping the mixture at high pressure into the column. The gradient is electronically controlled and programmable in
0.1 vol% increments. The injection valve has interchangeable sample loops, each of which holds a fixed volume.
Loops of different sizes hold volumes that range from 2 to 1000 µL. In the load position, a syringe is used to wash
and load the loop with fresh sample at atmospheric pressure. High-pressure flow from the pump to the column
passes through the segment of valve at the lower left.
The HPLC equipment uses steel or plastic columns that are 5–30 cm in length, with an inner diameter of 1–
5 mm. Columns are expensive and easily degraded by dust or particles in the sample or solvent and by irreversible
adsorption of impurities from the sample or solvent. Therefore, the entrance to the main column is protected by a
short guard column containing the same stationary phase as the main column. Fine particles and strongly adsorbed
solutes are retained in the guard column, which is periodically replaced. The most common HPLC column diameter
is 4.6 mm. There is a trend toward narrower columns for several reasons. Narrow columns are more compatible with
mass spectrometers, which require low solvent flow.
Most common detector in HPLC is refractive index detector. A refractive index detector responds to almost
every solute, but its detection limit is about 1 000 times poorer than that of the ultraviolet detector. The deflection-
type detector has two triangular 5-10 µL to compartments through which pure solvent or eluate passes. Collimated
(parallel) visible light filtered to remove infrared radiation (which would heat the sample) passes through the cell
with pure solvent in both compartments and is directed to a photodiode array by the deflection plate. When solute
with a different refractive index enters the cell, the beam is deflected, and different pixels of the array are irradiated.
Refractive index detectors are useless in gradient elution because it is impossible to match exactly the sample and
the reference while the solvent composition is changing. Refractive index detectors are sensitive to changes in
pressure and temperature. Because of their low sensitivity, refractive index detectors are not useful for trace
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analysis. Other detectors are spectrophotometric, electrochemical and evaporative light scattering detector (Harris,
2007).
Last component in the HPLC system is the readout system. The output will be recorded as series of
peaks—each one representing a compound in the mixture.
In the exercise, the following parameters of the HPLC system (normal-phase) was employed and 20 µL of
the test solutions were injected in the system. See Table 4.1.

Table 4.1. Parameters used in the HPLC system.

Parameters
Column Carbohydrate column
Solvent 80:20 CH3CN/H2O
Flow rate 2.0 ml/min
Detector Refractive index
Sensitivity 16x
Chart speed 2.5 cm/min
Test solution Sugar solutions

The assessment of the HPLC used was done by determining its efficiency, selectivity and resolution.
Efficiency of the column is reported as the number of theoretical plates. One way of determining the column
efficiency is the tangent method wherein it use the equation below
𝑁 = 16(𝑡𝑟 /𝑤)2
where tr is the retention time measured from the instant of injection and w is the peak width obtained by drawing
tangents to the sides of the Gaussian curve at the inflection points and extrapolating the tangents to intercept the
baseline. The distance between the intercepts is the peak width (Encyclopedia Britannica, 2018). There are other
ways to calculate efficiency namely 5σ method, half-peak method, etc. The tangent method is used to calculate the
efficiency of the column in reading the sugar standards in this experiment. The chromatograms of the standard sugar
used are shown in the following figures.

Figure 4.2. Chromatogram of fructose standard solution.

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Figure 4 4. Chromatogram of glucose standard solution.

Figure 3.4. Chromatogram of sucrose standard solution.

Figure 4.5. Chromatogram of xylose standard solution.

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The computed number of theoretical plates are summarized in the table below. Tangent method was used int the
calculation.

Table 4.2. Theoretical plates in the standard sugar solutions.

Sugar Retention time, Peak width at baseline, Number of theoretical plates (Tangent
min min method)
Fructose 4.007 0.5977011494 719.1019816
Glucose 4.440 0.643681609 761.2787745
Sucrose 6.065 0.6976744186 1209.138347
Xylose 3.442 0.5454545455 637.1249083

The plate number (N) is a measure of the peak dispersion on the HPLC column, which reflects the column
performance. Efficiency is derived from an analogy of Martyn and Synge who likened column efficiency to
fractional distillation, where the column is divided into Theoretical Plates. Each plate is the distance over which the
sample components achieve one equilibration between the stationary and mobile phase in the column. Therefore, the
more theoretical plates available within a column, the more equilibrations are possible, and the better quality the
separation.
The more plates (N) there are the narrower the distribution of carbon numbers from each trap (or plate).
Therefore, the higher the number of plates (N) the narrower the ‘peak’ obtained from that trap – this can be directly
related to the peak ‘efficiency’ in HPLC where a column with a high number of plates gives narrower, more
efficient, peaks (Crawford Scientific, n. d.). From the standards of US Food and Drug Administration, theoretical
plate should be greater than 2000 for the validation of chromatographic methods (ChromAcademy, n.d.). With this
limit, values obtained for number of theoretical plates does not match the FDA standards.
The height equivalent to a theoretical plate (HETP) can be calculated when both N and the column length
(L) are known
𝐻𝐸𝑇𝑃 = 𝐿⁄𝑁
As a result, the lower the HETP, the better the resolution and the more efficient the separation. Efficiency is
optimized when N is maximized and HETP is minimized. The van Deemter equation is another way of expressing
efficiency; it takes into account three factors that, along with the mobile phase linear velocity, will affect efficiency
and, therefore, the HETP value. These factors are represented by the variables A, B, and C in the van Deemter
equation
𝐻𝐸𝑇𝑃 = 𝐴 + (𝐵/𝜇) + 𝐶𝜇
in which A represents the Eddy diffusion phenomenon in the column, B represents molecular (or axial) diffusion, C
relates to the resistance to mass transfer, and μ is the linear velocity of the mobile phase through the
chromatographic system (Stauffer & Newman, 2008). In the experiment, the plate height, HETP, was not calculated
since the column length was unfortunately not recorded.
Another parameter evaluating the separation of the column is selectivity. Selectivity, α, describes the
relative retention of the components by the stationary phase or how well peaks are separated. The equation below is
used in calculating the selectivity.
𝛼 = (𝑡𝑟𝑥 − 𝑡𝑚 )/(𝑡𝑟𝑦 − 𝑡𝑚 ) = 𝐾2 /𝐾1
Where trx and try are retention times of component x and y and tm is the retention time of unretained species. The
variables K2 and K1 refer to the capacity factor. Capacity factor is a means of measuring the retention of the analyte
on the column. To determine the capacity/retention factor, K, the equation below is used.
𝐾 = (𝑡𝑟 − 𝑡𝑚 )/𝑡𝑚
To evaluate the capacity factor and selectivity of the system, chromatogram of ththe sugar standard solution
containing fructose, glucose and sucrose is used and the chromatogram for 50 mg/mL is shown below. as an
example. Peak A is fructose, Peak B is glucose and Peak C is sucrose. The peak identification was based on the
chromatogram of the individual sugar standard. The retention times of the peaks on Table 4.3.
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Figure 4.6. Chromatogram of 50 mg/mL sugar standard solution containing fructose, glucose and sucrose.

Table 4.3. Retention time and Peak widths of the peaks recorded standard sugar solution containing fructose,
glucose and sucrose.
Peak Retention time, min Peak width, min
10 mg/mL
A 4.008 0.3636363636
B 4.398 0.4545454545
C 6.018 0.4090909091
20 mg/mL
A 4.04 0.404494382
B 4.452 0.5168539326
C 6.093 0.6741573034
30 mg/mL
A 4.045 0.4318181818
B 4.48 0.5
C 6.158 0.5
40 mg/mL
A 4.085 0.4418604651
B 4.557 0.5581395349
C 6.2 0.6046511628
50 mg/mL
A 4.085 0.4318181818
B 4.552 0.5454545455
C 6.258 0.65

Retention time of the unretained species came from the chromatogram of distilled water and the value is 2.257
minutes. The chromatogram of distilled water is shown below.
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Figure 4.7. Chromatogram of distilled water.

Shown in Table 4.4 are the retention factors of the components of the solution. The largest gain in
resolution is achieved when the k value is between 1 and 5. As K is very simple to change it is often worthwhile
adjusting the k range of peaks within the chromatogram to obtain an optimum resolution k values less than 1 are
unreliable as analytes may be eluting with other sample components or solvent. Above a k value of approximately 5,
increasing retention only provides minimal increases in resolution. Too much retention wastes valuable analysis
time and the chromatographic peak height will decrease as the bandwidth of the peaks increases. For more complex
sample mixtures, the useful k range may be extended to 2 < k < 10. If you have not achieved the desired resolution
and the k values of your sample components are above 10, you will find that increasing the selectivity or efficiency
of your separation will be more useful (ChromAcademy, n. d.).
As seen in Table 4.4, peaks for fructose and glucose had values less than one except for the 50 mg/mL peak
for glucose and capacity factors for peaks of sucrose had values greater than 1. Fructose and glucose peaks had less
than 1 as value of K, thus, its separation is poor. Peaks of sucrose has more than 1thus, this value is acceptable for
good separation.

Table 4.4. Calculated capacity factors of the peaks recorded in 50 mg/mL standard sugar solution.
Concentration, mg/mL Peak A (Fructose) Peak B (Glucose) Peak C (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

Selectivity of the peaks are summarized in Table 4.5. High selectivity, α, values indicate good separating
power and a good separation between the apex of each peak. However, the alpha value is not directly indicative of
the resolution. By definition, the selectivity is always greater than one – as when α is equal to one, the two peaks are
co-eluting (i.e. their retention factor values are identical). The greater the selectivity value, the further apart the
apices of the two peaks become. As the selectivity of a separation is dependent upon the chemistry of the analyte,
mobile, and stationary phases all of these factors may be altered in order to change or optimize the selectivity of an
HPLC separation. Optimal α-values for most separations are between 2 and 5. As seen in the table below both
separations of fructose with glucose and glucose with sucrose had values lower than 2, thus not reaching the optimal
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values for selectivity. However, separation of Peak glucose with sucrose is better than separation of fructose with
glucose since selectivity values of former are higher in value.

Table 4.5. Calculated selectivity of the peaks recorded in standard sugar solution containing fructose, glucose and
sucrose.
Concentration, mg/mL α between fructose and glucose α between glucose and sucrose
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 resolution of the HPLC system is calculated using the equation

𝑅𝑠 = (1⁄4 (√𝑁)) 𝑥 (𝛼 − 1⁄𝛼) 𝑥 (𝐾 ⁄1 + 𝐾)
The most important thing in HPLC is to obtain the optimum resolution in the minimum time. A resolution value of
1.5 or greater between two peaks will ensure that the sample components are well (baseline) separated to a degree at
which the area or height of each peak may be accurately measured. The width at the base of each peak is the
segment of the peak base intercepted by the tangents drawn to the inflection points on either side of the peak as
shown. It should be noted that as resolution increases, so does the time require for the separation. As a
chromatographer, you will have to balance the desire for rugged separations with that of time and materials. The
fundamental resolution equation shown is affected by selectivity, efficiency and retention factor.
Resolution is a measure of ability of the column to resolve two solutes and alternatively it can be calculated
using the equation
𝑅𝑠 = 2(𝑡𝑟𝑥 − 𝑡𝑟𝑦) ⁄(𝑊𝑥 − 𝑊𝑦 )
The calculated resolution is summarized in the table below.
Table 4.6. Resolution of different concentrations of standard sugar solution containing fructose, glucose and sucrose.
Concentration, mg/mL Resolution between peaks 2 and 3 Resolution between peaks 3 and 4
10 0.9533333334 3.75157895
20 0.8943414634 3.0426875
30 0.9346585366 3.356
40 0.944 2.48196
50 0.9557209302 2.854144487

As seen in table 4.6, there is higher resolution in separation of glucose and sucrose than separation of fructose and
glucose. Evidently, the selectivity values show support on this conclusion.
Lastly done was quantification of sugar content of honey. Calibration curves of fructose, glucose and
sucrose were used in determining the quantities of sugar present in the honey sample. To construct the calibration
curve, peak area vs concentration was plotted. However, in the construction of the curve, data points from 50
mg/mL concentration was removed to achieved good linearity. Data on the calibration curve are summarized in
Table 4.7.

Table 4.7. Data on calibration curves of fructose, glucose and sucrose.

Concentration, mg/mL Peak area
Fructose Glucose Sucrose
10 541 1043 1377
20 2152 3218 2924
30 3468 4817 4417
40 4901 6802 6835
50 4261 6138 6114
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Slope 143.96 188.76 178.67

Y-int -833.5 -749 -578.5
R2 0.9984 0.9969 0.9853

The constructed calibration curves are shown in the following figure.

6000

5000 y = 143.96x - 833.5

R² = 0.9984
4000
Peak area

3000

2000

1000

0
0 10 20 30 40 50
Concentration, mg/mL

Fig 4.8. Calibration curve for fructose standard solution.

8000
7000 y = 188.76x - 749
R² = 0.9969
6000
5000
Peak Area

4000
3000
2000
1000
0
0 10 20 30 40 50
Concentration, mg/mL
Fig 4.9. Calibration curve of glucose standard solution.
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8000
7000 y = 178.67x - 578.5
6000 R² = 0.9853

5000
Peak Area

4000
3000
2000
1000
0
0 10 20 30 40 50
Concentration, mg/mL

Fig 4.10. Calibration curve of sucrose standard solution.

As shown in Table 4.8, the honey sample has 307.8285653 mg/mL, 260.2776012 mg/mL and 324.3689483
mg/mL fructose, glucose and sucrose respectively. Theoretically, honey is made up of around 75 per cent sugars, of
which roughly half is glucose and half is fructose (these proportions may vary depending on the source of the
nectar). The remaining 20 to 25 per cent is water with a trace of protein, a trace of fat and a trace of fibre, which
explains why honey has fewer ‘sugars’ or kilojoules/calories than sugar when you compare them weight for weight
(Saxelby, 2014).

Table 4.8. Concentration of fructose, glucose and sucrose in honey sample with dilution factor of 10.
Sample (DF = 10) Area Concentration, mg/mL
Fructose 3598 307.8285653
Glucose 4161 260.2776012
Sucrose 5217 324.3689483

IV. Conclusion
High Performance Liquid Chromatography was used in determining the quantity of fructose, sucrose and
glucose in honey sample. It was found out that the honey sample contains 307.8285653 mg/mL, 260.2776012
mg/mL and 324.3689483 mg/mL fructose, glucose and sucrose respectively.
Furthermore, in the experiment, the efficiency (number of theoretical plates), capacity factor, selectivity
and resolution of the column used were calculated. The number of theoretical plates of the reading of the standard
sugar solutions ranges from 600 to 1200. Higher number of plates means higher interaction with column, thus, better
separation. For capacity factor, peaks for fructose and glucose had values less than one except for the 50 mg/mL
peak for glucose and capacity factors for peaks of sucrose had values greater than 1. Fructose and glucose peaks had
less than 1 as value of K, thus, its separation is poor. Peaks of sucrose has more than 1thus, this value is acceptable
for good separation. For selectivity, optimal α-values for most separations are between 2 and 5. Both separations of
fructose with glucose and glucose with sucrose had values lower than 2, thus not reaching the optimal values for
selectivity. However, separation of Peak glucose with sucrose is better than separation of fructose with glucose since
selectivity values of former are higher in value. Lastly for resolution, there is higher resolution in separation of
glucose and sucrose than separation of fructose and glucose.
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V. References
Chromacademy. (n.a). The Theory of HPLC: Chromatographic Parameters. Crawford Scientific. Retrieved from
https://www.chromacademy.com/lms/sco2/Theory_Of_HPLC_Chromatographic_Parameters.pdf
Encyclopedia Brittanica. 2018. Efficiency and Resolution. Retrieved from
https://www.britannica.com/science/chromatography/Efficiency-and-resolution
Harris, D. (2007). Quantitative Chemical Analysis (7th edition). W.H. Freeman and Company. New York.
Saxelby, C. 2014. Food watch. Retrieved from https://foodwatch.com.au/blog/carbs-sugars-and-fibres/item/honey-
is-it-healthier-than-sugar.html
Stauffer, E. & Newman, R. 2008. Gas Chromatography and Gas Chromatography—Mass Spectrometry. Retrieved
from https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/theoretical-plate.