Experiment 6: High performance liquid chromatography (HPLC)

Theory/Background:

In recent years the technology used in chromatographic analysis has greatly improved.
Advances in this area have led to the common use of High Performance Liquid
Chromatography, a laboratory technique that allows for the every efficient separation of
small amounts of the components of a mixture. The technique has essentially the same
operational basis as liquid chromatography, but HPLC allows for far better separation of the
components of a mixture. The column in an HPLC instrument contains tiny particles of only
about 5 µm diameter, ensuring a very large surface area to which molecules may adsorb.
These particles comprise the stationary phase of the chromatographic system. Because these
tiny particles are so tightly packed, the mobile phase solvent must be forced through the
column under very high pressure. A detector and a computer are connected to the HPLC
instrument in order to signal when eluents are coming off the column and fractions should be
collected. The diagram in Figure 6 illustrates the main components of an HPLC instrument.

Identification of sample is carried out by comparison of their retention times. A known

amount of a known compound (a standard) is injected, the retention time is recorded and the
are under the peak can be calculated. A sample containing the same analyte is then injected
under the same experimental parameters. If the analyte is the same compound as the standard,
then they should give identical retention times. The ratio of peak areas should give the
amounts of the components present.

Reverse phase partition chromatography in HPLC employs a polar mobile phase and a non-
polar stationary phase. This is the most frequently used form of HPLC. Other modes of liquid
chromatography include normal phase partition (non-polar mobile phase and polar stationary
phase adsorption, ion exchange and size exclusion.

The column used in this experiment consists of a layer of an alkane C-18 chemically bonded
to the surface of very small silica particles. The alkane stationary phase is non-polar, the
silica is the inert support material with high surface area and the mobile phase is a polar
solvent (water/acetonitrile)

Objective:

-To determine the retention time of a standard solution (caffeine)

-To identify the caffeine peak in a soft drink sample

-To determine the amount of caffeine in soft drink sample using the response factor method

Apparatus:

Beaker

Burette

Glass rod

Volumetric flask 10mL

Dropper

Chemicals

Caffeine standard

Soft drink (coke/pepsi/100 plus)

Distilled water

Acetonitrile
Procedure:

A.Experimental

1. Caffeine Standard solutions

a. Stock solution 1.0mg/mL (solution A)
i. 10mg caffeine was weighed accurately into a 10mL volumetric flask
ii. It was dissolve in distilled water
iii. The volume was make up with distilled water.
b. Working solution,0.1 mg/mL (Solution B)
i. 1mL of solution A was pipetted into 10ml of volumetric flask
ii. The volume was make up with distilled water.
2. Sample preparation
a. 10mL of soft drink sample was poured into a small beaker
b. Magnetic stirrer was used to remove any carbonation
c. 2-5mL aliquot was filtered through a 0.45µm pore diameter membrane filter,
to remove particulate matter.
3. 10µL solution B was injected into the HPLC to establish the retention time, t R, of
caffeine
4. The peak area of caffeine was measured
5. 10µL was of prepared soft drink sample was injected into HPLC instrument
6. The peak area was measured corresponding to caffeine by referring to its tR.

B. Operation of the HPLC

Instruments

1. HPLC(UV DETECTOR) – PERKIN ELMER SERIES 200

2. HPLC (PDA DETECTOR) – SHIMADZU VP SERIES
Operation Instructions

Starting the instrument:

LC – 10 AT (liquid chromatography)

DGU – 14A (degasser)

CTO – 10AS (column/oven)

SPD – M10A(diode array detector)

SCL – 10A (system controller)

1. To operate the instrument, the system must be switch on, PC should be not switch
on first.
2. ON button was switched according to the labelled hardware number

Running the software:

1. To create method, go to class VP, click instrument 1

2. On login window,
a. Username: System
b. Password:2001
c. Click login
3. Click on pump icon
On pump window
Select mode: Low pressure gradient
Set up for flow: 0.2 ml/min
Pressure limit, max : 400psi
Min : 0psi
A: Buffer solution
B: Methanol(MeOH)
C: CAN(Acetonitrile)
D: H2O
4. Click CTO – 10 AS vp menu. Oven temperature : 28oC,T max:60oC
5. Click SCL -10 AV : Trigger type external
Power on ; Event 1
6. Go to status log menu: Do not change anything
7. Go to time program menu: Do not change anything
Click download
Click OK, then click apply
8. Select LC setup assistant icon: Click PDA image
9. In general menu, start wavelength :190nm, end wavelength:800nm
10. In library menu: Do not change anything
11. In purity menu: from 190nm to 800nm
12. In D/A output menu: Channel 1………………nm
13. In spectrum menu: Do not change anything
14. In multi menu: Insert appropriate data
15. In ratio menu
Channel 1
Wavelength (nm):_________
16. Click file > Method > Save as ……..
Description : (title)
17. Click LV setup assistant
18. Click turn on pump. At this moment, proceed with injection
19. Before injection taking place

Click control: simple run

Sample ID:…………..

Data file:……………

Click start

20. When filling the syringe with sample, the maximum volume should be 20µL and
you must quickly inject all sample into the port
21. Result will show a chromatogram with several peaks, depending upon sample
composition.
Sample preparation

1. Use syringe and a filter to transfer sample (10-20µL) into a vial

a. Type of filter: SRP 15 (0.45µm)
2. To prepare mobile phase, filter the mobile phase, for methanol, water, buffer and
acetonitrile (CAN), use membrane filter.
3. Use ultrasonic cleaner (Model: UC -05).
4. Sample injection
Click single run acquisition. Insert syringe and load
Inject sample and turn down.
5. Select control > extent run ________min
6. Select report > view > Method custom report.
7. Print results
8. For clean up process, click LC setup assistant
For mobile phase B,C and D, was with CAN. Click 100% CAN
For buffer, wash with water. Click 100% water
9. Switch off the instrument.
Results:

Compound Retention time, tR(min) Peak area of caffeine(%)

Caffeine standard 1.209 100%
Coke 1.212 35.34%
Coffee 1.219 32.03%

Report

1. Write a step-by-step operating procedure of the HPLC instrument you used. You may
draw block diagrams or use a flow chart.
2. Discuss the components and experimental conditions of the HPLC instrument you
used (mobile phase, normal/reversed phase, isocratic/gradient elution, detector, etc.)
3. Calculate the concentration of caffeine(ppm) in the unknown sample using the
following equations

Response Factor, RF, caffeine standard =

Peak area caffeine standard / Concentration of caffeine standard

Concentration of caffeine(ppm) in the unknown sample=

Peak area caffeine in sample / RF

(conversion factor:1mg/ml =1000ppm)

4. Include all the chromatograms obtained for submission with your reports. Indicate
which run was chosen to calculate the amount of caffeine in the soft drink and explain
why did you choose that run.
Questions

1. State the types of compounds which are suitable for analysis using HPLC
It should be unstable in terms of thermal and non-volatile such as organic, inorganic
biological samples, synthetic or natural polymers.

2. Differentiate between the HPLC and the GC in terms of:

a. Mobile phase
HPLC: Ultrapure water and liquid is made up of organic matter.
GC: Mobile phase is a gas and gases ion such as helium, nitrogen, argon or
hydrogen is to be analyzed (depend)
b. Column
HPLC: four-to-six inch long metal or glass tightly packed with silica or
differing carbon chain lengths
GC: coiled capillary columns with interior walls coated with various materials
and it can reach 100feets.
Discussion:

HPLC is one of the most powerful instrument for analytical chemistry, where it can separate,
identify and quantitate the compound that are present in any sample that can be dissolve in
liquid. There are lots of application for the instruments such as pharmaceuticals, food,
cosmetics, environmental matrices, forensic samples and industrial chemicals. The basic
component of HPLC are solvents (mobile phase), degassing system, pump, injector, column
and detector

Operating procedure of the HPLC instrument.

Components of the HPLC instrument

Solvent

The mobile phase in HPLC are mixture of polar and nonpolar liquid component. As
solvent passed through the column, as there is presence of contaminants, it could plug at the
column. This is why the solvent must be kept away from dissolves gases.

Injector

A syringe injection through self-sealing elastomeric septum. Most of the HPLC used a
glass Hamilton syringe that designed to withstand pressure up to 1500ps.
Column

Guard column used to prevent deactivation of the analytical column by adsorption and
minimize band broadening. Plus, it is used to increase the lifetime of HPLC column prevent
clogging of analytical column.

Detector

Located at end of the column, and is used to detect the presence of various component
of the sample, but it should not detect the solvent. The common detector that is used in HPLC
is UV absorption detector.

Techniques

One of the technique that is used in HPLC is degassing system technique. The
purpose of this technique is to remove dissolved gases and dust from liquid. As you know,
these interferences include bubbles may interfere the reading of most detectors. In this
experiment, it has been degassed using filtration through a membrane filter.

Normal phase vs Reverse phase

If stationary phase is more polar than mobile phase, it is said to be a normal phase, if
the mobile phase is more polar than stationary phase, it is called as reverse phase. In reverse
phase, the retention time of HPLC will increase as it decreased in polarity of particular
species. The compounds should be eluted at shorter time, which is better.

Gradient elution vs Isocratic elution

To know whether it is a gradient or isocratic elution, the composition of the mobile phase is
determined, whether it is constant or not. If the mobile phase is constant throughout HPLC
separation, it is said to be isocratic elution. If the compound was eluted while still
maintaining its peak resolution known as gradient elution. This technique was mostly chosen
when sample contains components of wide range of polarities. The gradient elution offers
most complete separation of peaks in shorter time period without loss of resolution in earlier
peaks
Concentration of caffeine (ppm) in the Coke and Coffee sample

RF= 100 / 1mg/mL

=1000mg/mL

Concentration of caffeine(ppm) in coke sample

35.34 / 1000

=0.03534 x (1000ppm / 1mg/mL)

=35.34ppm

Concentration of caffeine(ppm) in coke sample

32.03 / 1000

=0.03203 x (1000ppm / 1mg/mL) = 32.03 ppm

Chromatogram obtained in the experiment

Each of the sample, mostly run for five time, to get an accurate results, means that there are
15resultscombination of coke, coffee and caffeine chromatogram. The chromatogram that
have nearest value to standard caffeine ,which is 1.209 is chosen, to calculate the amount of
caffeine presence in the coke and coffee. The chromatogram with the lowest retention time
give the best result.

Conclusion:

In the nutshell, the retention time for the standard solution which was caffeine is
1.209. Correlate with that, by using response factor method, the peak of the caffeine in soft
drink was determined which was 1000 mg/mL.
References:

(Ghiotti & Boccuzzi, 1987; Ishii, Tsuboi, Sakane, Yamashita, & Breedlove, 2009; Kalmus &
Harper, 1915; Kumar, Kumar, & Dass, 2013; Nitschké, Ertl, & Küppers, 1981)

Ghiotti, G., & Boccuzzi, F. (1987). Chemical and Physical Properties of Copper-Based
Catalysts for CO Shift Reaction and Methanol Synthesis. Catalysis Reviews, 29(2–3),
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Ishii, T., Tsuboi, S., Sakane, G., Yamashita, M., & Breedlove, B. K. (2009). Universal
spectrochemical series of six-coordinate octahedral metal complexes for modifying the
ligand field splitting. Dalton Trans., 0(4), 680–687. https://doi.org/10.1039/B810590A

Kalmus, H. T., & Harper, C. (1915). Physical Properties of the Metal Cobalt. Journal of
Industrial & Engineering Chemistry, 7(1), 6–17. https://doi.org/10.1021/ie50073a004

Kumar, D., Kumar, A., & Dass, D. (2013). Syntheses and Characterization of the
Coordination Compounds of N-(2-hydroxymethylphenyl)-C-(3′-carboxy-2′-
hydroxyphenyl)thiazolidin-4-one. International Journal of Inorganic Chemistry, 2013,
1–6. https://doi.org/10.1155/2013/524179

Nitschké, F., Ertl, G., & Küppers, J. (1981). Coordination chemistry of metal surfaces:
Chemisorption of PF 3. The Journal of Chemical Physics, 74(10), 5911–5921.
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