Microsoft Word - Analytical practicals Manual
17
Experiment 8: Separation of Additives in Soft Drinks by HPLC using a
XXXXXXXXXXNon-polar Column with Polar Endcapping.
The challenge of HPLC is to obtain retention of hydrophilic components in samples.
The more commonly used C8 or C18 columns (reverse phase) do not always provide
the selectivity necessary to retain such hydrophilic (polar) compounds in samples.
Note that there is no such difficulty with the more non-polar components in a mixture.
The introduction of HPLC columns containing polar endcapping and/or polar
embedded groups allows for the retention of these hydrophilic compounds. The
conditions described in this practical allow the separation of additives commonly
found in soft drinks that cannot be separated on conventional alkyl-bonded phases
(C18).
The major problem in analysing soft-drink additives is retaining the highly polar
components while keeping the retention of the hydrophobic analytes down to an
acceptable k’ range. Because of its highly polar selectivity, asco
ic acid is virtually
unretained using C18 or C8 columns when using conditions that elute the more
hydrophobic components within a working k’ range. Using the Synergi Polar RP
column the asco
ic acid is retained beyond the void volume while the last eluted
peak has a k’ value of approx. 5.
Column: Synergi 4μm Polar-RP 80A (150 x 4.6 mm)
Synergi is based on a new 80A ultra-high purity (> 99.9% metal free), base
deactivated type silica, which ensures minimal surface metal sites available for
chelation and reduced silanol (Si-OH) acidity even at neutral pH. The high metal
content of older type silicas can result in poor recovery or adsorption of certain
analytes due to chelation and contribute to peak tailing of basic compounds.
4μm generates columns with efficiency intermediate between 3 or 5μm and with a
pressure drops similar to those obtained with columns packed with 5μm particles.
(unexpected – should be higher – this unexpected bonus is due to the different type
silica)
80A Pore Size for High Surface Area: The high (475m2/g) surface area results from
a unique 80A pore geometry. This increases sample analyte interaction with the
onded phase and it also compensates for the reduced hydrophobicity of Synergi 4μ
Polar-RP, allowing it to retain both hydrophobic and hydrophilic analytes through
non-polar and polar interactions respectively.
Synergi-Polar-RP: Is an ether linked phenyl phase with proprietary hydrophilic
endcapping designed specifically to maximise retention and selectivity for polar,
aromatic analytes that are so often encountered in the pharmaceutical industry Due to
its bonding chemistry, Syn. Polar-RP displays a polar selectivity that complements the
more conventional selectivity of Syn. Max-RP. The ether – linkage not only improves
selectivity for acidic and basic compounds but also gives high aqueous mobile phase
stability. Eg. Methanoic acid can be separated on an embedded basic group (N -
containing amide link) on a non-polar column. The basic group however can interfere
with the resolution of highly , polar compounds. However since the SP used here
uses an ether linkage as the embedded polar group, the result is improved peak shape
and separation of highly acidic, polar analytes. In addition this SP’s ether linkage is
18
extremely resistant to hydrolysis even at pH 1.5, thus enabling separations under
elatively harsh conditions. At the other end of the pH spectrum, it is stable to pH 7.0.
Mobile Phase 55 of .02M KH2PO4 adjusted to pH 2.3 then mixed with: 45 of
Methanol @ a flow rate of 1.0cm3/min.
Detector: UV detector at 220nm. (Preferably Diode A
ay).
Procedure:
1. Stock solutions of the following standards are provided with concentrations of
100mg/100cm3:
Asco
ic Acid; Acesulfame K; Saccharin; Aspartame; So
ate; Benzoate;
Caffeine. The compounds elute in this order from the RP-Polar column..
2. Using the stocks provided make up standards of concentration 1, 2, 3, 4, & 5
mg/100cm3 in 100cm3 volumetric flasks. Use the mobile phase as solvent.
One Exception: Take 5, 10, 15, 20 & 25cm3 of the Asco
ic acid solution.
3. Samples: Take 25cm3 of each of three drinks and dilute to 100cm3 with MP.
Important Note: If you do not obtain a peak for aspartame in Lilt and Energise
Sport you will have to inject undiluted samples of these to measure the amount
of aspartame in these drinks.
4. Analyse each of the 8 solutions on the RP-Polar column and determine the % of
each component in each of the three drinks. (Can be 10 injections – See 3 above)
5. Meanwhile take 1.0cm3 (approx) of each of the stocks and dilute each one
separately to 10cm3in eight 10cm3 volumetric flasks. Analyse these on a 150mm C18
column on a separate HPLC using the same mobile phase. Report their retention times
and compare with your results from the other HPLC.
Compound tr on RP-Polar using
45:55 Methanol:KH2PO4
tr on C18 using
45:55
Methanol:KH2PO4
Asco
ic Acid
Acesulfame K
Saccharin
Aspartame
So
ate
Benzoate
Caffeine
19
6. Draw the appropriate graphs and determine the % of each component in each drink.
Asco
ic Acid ____%; Acesulfame ____%; Saccharin ____%; Aspartame ____%;
So
ate ____%; Benzoate ____%; Caffeine____%;
7. Look up the structures of each component and rationalise the tr values with the
structure, the mobile phase used and the stationary phase.
8. Comment on the difference between the tr values under the two sets of experimental
conditions. Could the C18 column and its mobile phase be used? Give the reasons for
the differences between the two sets of tr values.
9. Read the article “The Benefits of Reversed Phases with Extended Polar
Selectivity in Analysing Wide-Polarity – Range Samples” in LC-GC March 2002
pages 156 –164. This will help you to answer the above questions.
Synergi Polar Reverse Phase Column: Increased retention of highly polar and
aromatic compounds
Highly reproducible and stable phenyl phase
Enhanced polar resolution in 100 % buffer mobile phases
Peak area
STDs
mg/100cm3 STD 1 STD 2 STD 3 STD 4 STD 5
Asco
ic acid XXXXXXXXXX XXXXXXXXXX
Acesulfame K XXXXXXXXXX XXXXXXXXXX
Saccahin XXXXXXXXXX XXXXXXXXXX
Aspartame XXXXXXXXXX 9818
So
ate XXXXXXXXXX XXXXXXXXXX
Benzoate XXXXXXXXXX XXXXXXXXXX
Caffeine XXXXXXXXXX XXXXXXXXXX
Energise
sport Lilt Red bull
Asco
ic acid 24324
Asco
ic
acid 3235
Asco
ic
acid 3235
Acesulfame K 20544
Acesulfame
K 1515
Acesulfame
K 1515
Saccahin
not
detected Saccahin 928 Saccahin
not
detected
Aspartame 1860 Aspartame 2270 Aspartame 2270
So
ate 17363 So
ate 2016 So
ate
not
detected
Benzoate 55001 Benzoate 6434 Benzoate 2029
Caffeine
not
detected Caffeine
not
detected Caffeine 236116
LC•GC Europe December 20022
COLUMN WATCH
Reversed-phase chromatography is by fa
the most widely used mode in high
performance liquid chromatography
(HPLC).1 It allows chromatographers to
manipulate the mobile phase by changing
organic solvent type, solvent composition
and pH; by adding modifiers such as
surfactants, chiral reagents, competing
ases and ion-pair reagents; or by
adjusting experimental conditions such as
flow-rate and temperature. Originally,
esearchers believed that they would need
only a few stationary phases to achieve
virtually any separation they encountered.
Indeed with a C18 column and the ability
to adjust the myriad parameters,
chromatographers have achieved many
successful separations. Still, sometimes
analysts can neither find the necessary
selectivity nor obtain a rugged and
eproducible separation easily, no matte
how many parameters they adjust.
Early Modified Surfaces in
Reversed-Phase Chromatography
New and modified reversed-phase
chromatography stationary phases have
een introduced throughout the years to
provide more separation power. In
eversed-phase chromatography, basic
compounds can frequently interact with
unreacted silanols on the silica gel because
it is virtually impossible, at least with
monomeric bonding, to remove or cover all
of the silanols because of steric reasons,
especially with long alkyl chain phases such
as octadecylsilane (C18). This interaction is
most problematic when the packing is used
at intermediate pH values of pH 4–8 at
which silanols and many basic compounds
are partially ionized.
Twenty years ago, the low-purity silica
gel used for most HPLC columns caused
high surface acidity and a tailing problem
with basic compounds. The first modified
stationary phases were the so-called base-
deactivated bonded silicas. These base-
deactivated phases were not deactivated
with base, but the term implied that the
column had been treated to provide
minimal interaction and tailing with
strongly basic compounds