Analysis of Limonin in orange juice by HPLC (1)
Maria Matyska-Pesek, Joseph Pesek
Chemistry Department, San Jose State University, San Jose, California, USA
Overview: HPLC – High Performance Liquid Chromatography.
This experiment uses high performance liquid chromatography (HPLC) to determine the
concentrations of limonin in orange juice.
Equipment:
1. HPLC system with a diode array detector (UV) which will be used for limonin
determination.
2. Cogent C18 column, 4.6x100mm, 4 m particle size (MicroSolv Tech. Corp.
Eatontown, New Jersey, US)
Purpose: Limonin is a terpenoid compound that is found in various citrus juices. In the
citrus fruit itself however, only the limonin precursor limonate-A-ring lactone (LARL) is
present. When the juice is extracted, the seeds that contain LARL are broken and mix
with the rest of the acidic fruit matrix. This catalyzes a conversion of LARL to limonin.
The significance to the citrus fruit industry is that while LARL is tasteless, limonin is
primarily responsible for bitterness in these juice products. Another factor to consider is
that in the intact fruit, LARL is slowly converted via limonoid glucosyltransferase into
another tasteless compound, limonin 17-β-D-glucopyranoside. Hence, low LARL levels
roughly estimate fruit maturity, since less LARL will be available to convert to limonin in
the juicing steps. Please find the structures of limonin, LARL, and limonin 17-β-Dglucopyranoside and upload them to your lab notebook. These compounds and other
liminoid related species can be found in many citrus products.
After analyzing the juice samples, it is then possible to determine the concentration of
limonin in the orange juice tested and evaluate the influence of the OJ matrix on the
analysis (2).
A.
Equipment and chemicals




Agilent 1050 or similar HPLC system with a diode array detector, computer
and an Autosampler (1 -100 L)
Column: Cogent Type-C BDC-18, 4.6 x100 mm, 4 m particle size),
MicroSolv Tech Corp. Eatontown, New Jersey. US
printer
mobile phase bottles (2 bottles):
A: DI water+0.1% formic acid,
B: Acetonitrile+0.1% formic acid,
Mobile phase used is isocratic: 65% A/ 35% B and it will be delivered by the
1



B.
C.
pump at flow rate: 0.5 mL/min
Orange juice
screw-capped sample vials for HPLC instrument used, 12 vials, per group of
four students - 3 per student are needed.
45 m nylon filters and syringes
Additional items (One per group of four students)
 One 1000 L (microL) micropipette with tips
 one 1-100 L (microL) micropipette with tips
 four 50 mL beakers (one per student)
 four weighing bottles with caps (clean and dry) – one per student
PROCEDURES
C.1 PREPARATION OF THE UNKNOWN SAMPLE
C.1.1.
A. Place eight sample vials in the vial holder.
B. Clean weighing bottles with water and soap, triple rinse them with DI water and place
them in the oven to dry.
C.1.2. Bring a 50 mL marked (with your name) beaker to the lab instructor for your
unknown sample (orange juice).
C.1.3. EACH STUDENT PREPARES HIS/HER OJ SAMPLE
When the weighing bottles are dry, use one of them for the filtered sample. Prepare the
filtered sample as follows: using the syringe withdraw a small portion of the OJ (~0.5
mL) and rinse the syringe tip. Discard rinse in the sink. Repeat two more times, but this
time filtering the solution into the weighing bottle (use 0.45 m nylon syringe filters
provided), rinse the weighing bottle with the filtered sample solution. Discard the rinse
to the sink. Filter about 2 to 5 mL of the orange juice into the weighing bottle and cap
promptly. Transfer about 400 L of the filtered OJ sample solution into the Autosampler
vial using a micropipette (see NOTE 1 below on how to use micropipette). THIS IS
YOUR UNKNOWN OJ SAMPLE. Mark the vial as “Unk.OJ” and your name.
Save the rest of the filtered OJ sample for preparation of the spiked sample.
Alternative sample preparation:
If the filtering is too difficult centrifuge OJ sample at 3,000 g for 10 min using glass tubes
provided in the centrifuge (clean and dry them before use). After 10 min of centrifuging,
filter the supernatant (about 400 L) through a 0.45 µm nylon syringe filter into an
Autosampler vial. NOW THIS IS YOUR UNKNOWN SAMPLE “Unk.OJ”. Filter the rest
of the supernatant into the clean and dry weighing bottle. Save the rest of the filtered OJ
sample for preparation of the spiked sample.
The filtering of any sample has to be done before injecting it into the HPLC column.
Otherwise, solids present in the sample will clog the analytical column, which is very
expensive.
2
PREPARATION OF STANDARD ADDITION SAMPLE
C.1.4.
A. Using a micropipette, transfer precisely 1.00 mL of the filtered juice (prepared above)
into an HPLC vial. Using micropipette (1-100L) add 200 L of limonin standard (obtain
limonin standard from the instructor) to the same vial. Record the concentration of the
limonin standard. Cap and mix thoroughly. Mark this vial as “Spiked OJ”.
See NOTE 1 below how to use a micropipette)
Please remember to tightly screw the septum cap of vial and mix the contents by
shaking the vials. (These are the standard addition, samples – one per student.)
B. Using a micropipette, transfer precisely 1.00 mL of DI water into an HPLC vial. Using
a micropipette (1-100L) add 200 L of limonin standard (obtain limonin standard from
the instructor) to the same vial. Record the concentration of the limonin standard. Cap
and mix thoroughly. Mark this vial as “Limonin”. Calculate the concentration of
limonin in the vial.
See NOTE 1 below how to use micropipette.
Please remember to tightly screw the septum cap of vial and mix the contents by
shaking the vials. (These are the standard samples – one per student.)
In the end of the above sample preparation each student will have 3 vials (unknown OJ,
Spiked OJ and Limonin).
Place the prepared samples in order in the Autosampler of HPLC system.
Record placement of your samples.
Instructions on how to operate the HPLC instrument will be given by the instructor. The
instructor will also help you set up a sequence for injecting the samples.
After all runs are done you can print or analyze the data using the “offline” instrument
(next lab period).
The “offline” program is used to analyze data, create new methods and sequences.
The use of the HPLC instrument will be demonstrated by the instructor.
3
NOTE 1:
Procedure for using the micropipette:
Set the micropipette to 1.000 mL and attach a disposable pipet-tip. Wet the pipet-tip by
immersing it 2-3 mm into the filtered sample solution; depress the control button to the
first stop and then slowly release the button (draw solution in), repeat the fill-and-expel
three times; then, with the tip out of solution, depress the button to the second stop to
completely empty the tip after the third wetting cycle. Now to fill the tip with exactly
1.000 mL, depress button to the first stop; re-immerse the tip (2-3 mm) in the filtered
sample solution (in the weighing bottle) and allow the button to glide back slowly. Slide
the tip out along the inside of the vial. Wipe off any droplets clinging to the exterior of the
tip with lint-free tissue (Kimwipe). (Do not wipe close to the pipet-tip opening to avoid
absorbing out any of the sample contents.) To dispense the 1.00 mL standard solution
into an autosampler vial, place the pipet-tip against the dry inside surface of the vial;
press the control button slowly down to the first stop and wait 1-3 sec; then continue to
press the button down to the second stop; this will deliver all remaining liquid in the
pipette tip. While continuing to hold the control button down, slide the tip out along the
inside of the vessel. When completely removed eject the pipet-tip by depressing the
control button to the final stop. Tightly screw the septum cap of the vial.
D.
Data Analysis
Looking at the chromatogram of the limonin standard, assign the peak for limonin in the
unknown OJ and spiked OJ samples. The peak area (in A.U.*min, units) of most peaks
is printed in the integrator section of the printout. Record the peak area of the limonin
4
peak in the standard sample, OJ unknown sample and in the spiked OJ sample.
Calculate limonin concentration in HPLC vial marked “limonin std”. Calculate the
concentration of limonin in the unknown OJ sample by the standard addition method.
Calculate the number of theoretical plates (N) for limonin in the “OJ spiked” sample.
Record results in your lab notebook and on the HPLC report sheet.
Note:
In the chromatogram of OJ and spiked OJ you will notice additional small peaks. These
are impurities or other compounds present in the juice matrix.
Please remember to upload relevant chromatograms (not all chromatograms) into your
electronic lab notebook.
E. Calculations for Standard Addition Method
B = before spiking (unknown sample)
C = after spiking
Note: Use equation 3 to calculate CB (number you report).
For constant conditions of sample size (injection volume), flow rate, solvent composition,
and column conditions, the area of the peak corresponding to the target analyte is
proportional to its concentration in the injected sample mixture.
AB = kCB
(1)
where AB = the target analyte (in the unknown sample) peak area and CB = target analyte
concentration in the sample solution Unk. (unknown sample); and k is the proportionality
constant for the specified experimental conditions. The peak area, AC, for the sample plus
standard addition (spiked OJ sample), can be related to the added quantity of analyte:
AC = kCC = k[CBVB + CSV]/(VB+V)
(2)
where, CC is the concentration of sample-plusstandard; VB is the original sample volume (1.00
mL) containing the unknown analyte; CS and V
are, respectively, the concentration (printed on
the bottle with the standard) and volume (200L)
of added standard. Thus, by dividing Equation (2)
by Equation (1), and solving for CB:
Solution B (Sample)
Retention
time
1
3
t
M
2
Solution C. With Std Addn to second peak.
CB = (ABCSV)/[AC(VB+V) - ABVB]
(3)
Retention
1
time
t
The concentration of analyte in the original
sample, CB, can be computed from Equation (3),
if the injection volumes are identical for the two
chromatograms.
2
3
M
Lcstad2.ch3
Figure 1.Standard Addition of standard
(limonin). Upper trace is for solution Unk
OJ; lower trace is “spiked OJ.
5
F. Determination of Capacity Factor (k)
Find out t0 value from the instructor
G. Determination of Number of Theoretical Plates
Use the chromatograms printed out. To your unknown peak, draw tangent lines at the
inflection points of the peak, which are indicated by the first and the third dotted lines of
each peak. Extrapolate the tangent lines to intersect the baseline. The width of the peak
(w0.1) is obtained by measuring the corresponding time difference between the first and the
second tangent-baseline intersections. To determine N0.1, use the equation below, with tR
and w0.1 measured in the same units (min. or sec.).
Figure: Determination of “N”, Theoretical Plate Number, HPLC.
Use the printout of enlarged peak of your analyte (in the spiked sample) to calculate w0.1
value.
6
For your information there are 3 more methods to calculate N0.1
Half Peak Height Method is second most commonly used method
In this method width is calculated from the width at half the peak height (w0.5). Since
width can be calculated easily by hand, it is the most widely used method. This is the
method used by the DAB (German Pharmacopeia), BP (British Pharmacopeia), and EP
(European Pharmacopeia). The coefficient in the equation is then 5.54 instead of 16 as
in Tangent Line method
The Japanese Pharmacopoeia 15th revision issued in April 2006 changed the
coefficient from 5.55 to 5.54.
Literature:
1. QUANTITATIVE ANALYSIS LABORATORY Manual, SAN JOSE STATE
UNIVERSITY, Spring 2015, Compiled by: Prof. Joseph J. Pesek, Dr. M.T.
Matyska-Pesek, Contributors: J. Neptune, R. Terrill and M. Stephens
2. "LC-MS Compatible Approaches for Quantitation of Limonin in Citrus Juice.",J.E.
Young, J.J. Pesek, M.T. Matyska,, LCGC, 33 (2015) 192-199.
7
Results and Calculations
HPLC Determination of Limonin in orange juice
Name
Section
Date
Locker No.
Partners' Names
Instrument System
(Mfr., Model No., I.D.)
SAMPLE
Unknown OJ
Limonin Standard
Limonin (210 nm)
Compound:
Retention Time, Sec.
Peak Width (w0.1), Sec.
Limonin (210 nm)
sec.
sec.
sec.
sec.
Theor. Plates, N0.1
Capacity Factor, k
Standard concentration (ppm)
Mobile
Phase
ppm
Experimental Conditions
Column
Flow
Temp.
Material
Rate
Deg. C.
Sample Preparation
Sample Volume (mL)
Std. Vol. Added (microL*.001)
File
Name
Injection
Vol, microL
Volume (mL)
mL V B
mL  V
Limonin peak (210 nm)
Limonin/sample
Peak area (AU*min.)
BEFORE Std. Addition (unknown OJ)
AFTER Std. Addition (spiked OJ)
Calculated Analyte Concentration (ppm)
in orange juice
AB
AC
ppm
Please remember to convert units for AB and AC from the printout to units required in this report.
8
HPLC Determination of Limonin in orange juice
Report Page
(SAMPLE)
Name
Anna Bajor
Section
O3
Date
03/24/15
Locker No.
42
Partners' Names
Joe,Kasia, Jurek
Instrument System
Agilent 1090 HPLC System
(Mfr., Model No., I.D.)
DH 413B, SN:1039764598
SAMPLE
Unknown OJ
Limonin Standard
Theor. Plates, N0.1
Limonin (210 nm)
138s
21s
700
Limonin (210 nm)
142s
22s
700
Capacity Factor, k
1.55
1.62
50 ppm
Compound:
Retention Time, Sec.
Peak Width (w0.1), Sec.
Standard concentration (ppm) 300 ppm
Mobile
Phase
Experimental Conditions
Column
Flow
Temp.
Material
Rate
Deg. C.
65% DI water+0.1%Formic acid
25 0 C
0.5 mL/min
Blue
35% acetonitrile+0.1% formic acid
Sample Preparation
Sample Volume (mL)
Std. Vol. Added (microL*.001)
Volume (mL)
Vb
1.0 mL
V
0.2 mL
Limonin peak (210 nm)
Limonin/sample
Peak area (AU*min.)
BEFORE Std. Addition (unknown OJ) 0.0569
AFTER Std. Addition (spiked OJ)
0.0845
Calculated Analyte Concentration
(ppm) in orange juice
33.6ppm
9
File
Name
AB
AC
Injection
Vol, microL
5 L
10