Technical Note 63
Peak Identification and Estimation of Percent
Purity using HPAE with 3-D Amperometry
INTRODUCTION
High Performance Anion-Exchange chromatography with Pulsed Amperometric Detection (HPAEPAD) or with Integrated Pulsed Amperometric Detection (IPAD) are established techniques1 for carbohydrate
and amino acid analysis, respectively. These techniques
have proven capabilities for separation of amino acids
and carbohydrates in complex samples containing a
large number of ingredients such as fermentation broths
and cell culture media.2–7 The highly sensitive direct
detection of amperometry eliminates the need for preor post-column derivatization. Chemical derivatization
techniques complicate analysis, add cost for expensive
reagents, introduce safety hazards to lab personnel
exposed to toxic solvents, and add a hazardous waste
stream that must be safely disposed of. HPAE with
amperometric detection eliminates these complications. The high sensitivity of amperometric detection
enables determinations of high femtomole amounts, and
routinely to low picomole amounts, of amino acids and
carbohydrates.
Amperometry is an electrochemical detection method that utilizes a flow cell consisting of three electrodes
(working electrode, reference electrode, and counter
(auxiliary) electrode).
Varying voltage potentials are applied between
the working electrode and the reference electrode by
means of a waveform program stored in the detector
software. The waveform program typically runs for less
than 1 second, repeating its program cycle throughout
the chromatographic period. Applying defined voltages allows specific analytes separated by the analyti-
cal column to be oxidized. The oxidation of the analyte
results in a flow of electrons (current) to the working
electrode surface. The current is measured between the
working and counter electrodes and integrated during a
defined time period within the waveform to yield charge
(coulombs) and then recorded by the data management
system (i.e., Chromeleon®). Certain compounds can be
oxidized at a given applied voltage potential, and therefore amperometry can impart a degree of specificity by
adjusting the applied voltage.8 3-D amperometry extends
conventional amperometry by enabling the continuous
acquisition of measured current throughout the entire
waveform period rather than just a pre-defined period
within the waveform when current is integrated. Having
the complete data set allows, among other things, postchromatographic current integration. Because different
chemical compounds oxidize differently at given applied
voltages, subtle differences in the amount of current
generated through a waveform can provide additional information about the identity and purity of the substances
being analyzed. These differences can be measured by
comparing the peak areas obtained for current integrated
through different time periods within the waveform. The
ratio of peak areas from different integration periods for
a given analyte allows comparison of unknown and standard peaks. An unknown peak may be identified when
its peak area ratio is numerically the same as that of the
standard (having the same retention time). The extent
of deviation between these ratios is a measure of the
extent of purity. The relationship of 3-D amperometry
to conventional amperometry is in some ways similar to
the relationship that diode array detection has to single
wavelength UV absorbance detection.
In this technical note, we present detailed instructions for the procedure used to obtain 3-D amperometry
data and extract useful information possibly leading to
identification of peaks and estimation of their extent
of purity. In a previously published article,6 measured
leucine (Leu) concentrations of a cell culture medium
exceeded the concentrations described by the manufacturer. Here, the application of 3-D amperometry
determined that the presupposed Leu peak was actually
another ingredient in the media, coeluting with leucine.
After identifying HEPES as the coeluting substance, 3-D
amperometry was used to estimate the concentration of
Leu coeluting with HEPES. Although 3-D amperometry
cannot be used for identification and estimation of impurity concentrations in all applications, this technical note
describes the procedures required to evaluate whether
3-D amperometry can be used for peak identification and
estimation of purity for a given application.
EQUIPMENT
Dionex ICS-3000 system consisting of:
DP-1 Gradient Pump (optimized for 2 mm i.d. columns), with in-line degas option.
DC-1 Electrochemical Detector and compartment
Consisting of combination pH/Ag/AgCl reference
electrode and AAA-Certified™ Disposable Au
Working Electrode (P/N 060082 for pack of 6; P/N 060140 for 4 bundled packages) or
AAA-Certified Au Working Electrode (non-disposable, P/N 063722)
AS Autosampler
Chromeleon Chromatography Management Software
CONDITIONS
AAA-Direct ™ Method:
Columns:
AminoPac® PA10 Analytical (P/N 55406)
AminoPac PA10 Guard (P/N 55407)
Eluent:
A: 10 mM NaOH
C: 25 mM NaOH/1 M Sodium acetate
Flow Rates: 0.25 mL/min
B: 250 mM NaOH
D: 100 mM Acetic acid
Inj. Volume: 25 µL
Temperature: 30 °C
Detection:
Gradient
Methods:
Integrated pulsed amperometry, disposable
or conventional Au working electrodes
See Table 1
Note: In this document, gradient methods are
described as x/y, where x is the initial NaOH eluent
concentration, and y is the isocratic time for this eluent.
For example, method 15/8 refers to the program method
using 15 mM NaOH as the starting eluent concentration, and it is held for 8 min before the start of the NaOH
gradient.
Waveform
Potential (V)
Time(s) vs pH Gain Region
0.00
0.04
0.05
0.21
0.22
0.46
0.47
0.56
0.57
0.58
0.59
0.60
+0.13
+0.13
+0.33
+0.33
+0.55
+0.55
+0.33
+0.33
-1.67 -1.67
+0.93
+0.13
Off
Off
Off
On
On
On
On
Off
Off
Off
Off
Off
Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry
Ramp Integration
On
On
On
On
On
On
On
On
On
On
On
On
Off
Off
Off
On
On
On
On
Off
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Table 1. Modified AAA-Direct Gradient Methods
Initial NaOH Eluent Concentration:
Initial Isocratic Time: AAA-Direct Method Name: Event
Time
(min)
Curve
Type
%A
10 mM
NaOH
0.0
5
100–%B
8.0
5
100–%B
14.0
8
66.7
17.0
5
66.7
24.0
8
27.0
30.0
REAGENTS AND STANDARDS
Reagents
Sodium hydroxide, 50% (w/w) (Fisher Scientific and
J. T. Baker)
X
8 min
X/8
Deionized water, 18 MΩ-cm resistance or higher
%B
250 mM
NaOH
%C
25 mM
NaOH +
1M
Sodium
Acetate
%D
100 mM
Acetic
Acid
Sodium acetate, anhydrous (AAA-Direct Certified,
Dionex Corporation, P/N 059326)
Acetic acid, glacial (17.5 M; HPLC grade, 99.7%
minimum; J. T. Baker)
Standards
Amino acid standard mix (NIST, Standard Reference
Material 2389)
*
0.0
0.0
*
0.0
0.0
33.3
0.0
0.0
33.3
0.0
0.0
1.0
89.0
10.0
0.0
5
1.0
89.0
10.0
0.0
8
0.0
80.0
20.0
0.0
32.0
5
0.0
80.0
20.0
0.0
34.0
8
40.0
30.0
30.0
0.0
36.0
5
40.0
30.0
30.0
0.0
38.0
8
30.0
30.0
40.0
0.0
PREPARATION OF SOLUTIONS AND REAGENTS
40.0
5
30.0
30.0
40.0
0.0
Sodium Hydroxide Eluents
42.0
8
20.0
30.0
50.0
0.0
44.0
5
20.0
30.0
50.0
0.0
46.0
8
10.0
30.0
60.0
0.0
48.0
5
10.0
30.0
60.0
0.0
50.0
8
0.0
30.0
70.0
0.0
62.0
5
0.0
30.0
70.0
0.0
62.1
8
0.0
0.0
0.0
100.0
64.1
5
0.0
0.0
0.0
100.0
64.2
8
20.0
80
0.0
0.0
66.2
5
20.0
80
0.0
0.0
66.3
5
100–%B
*
0.0
0.0
92.0
5
100–%B
*
0.0
0.0
10 mM and 250 mM Sodium hydroxide
It is essential to use high-quality water of high
resistivity (18 MΩ-cm). All water is filtered through
a 0.2-µm nylon filter (Nalgene 90-mm Media-Plus,
P/N 164-0020; Nalge Nunc International) under vacuum
to degas. Biological contamination should be absent. It
is important to minimize contamination by carbonate, a
divalent anion at high pH that is a strongly eluting species that causes changes in amino acid and carbohydrate
retention times. Commercially available NaOH pellets
are covered with a thin layer of sodium carbonate and
should not be used. A 50% (w/w) NaOH solution is
much lower in carbonate (carbonate precipitates in 50%
NaOH) and is the required source of NaOH.
Dilute 26 mL of 50% (w/w) NaOH solution into
1974 mL of thoroughly degassed water to yield 250 mM
NaOH. Dilute 1.05 mL 50% NaOH into 1999 mL water
to yield 10 mM NaOH. Immediately blanket the NaOH
eluents under 4–5 psi helium or nitrogen to reduce carbonate contamination.
* To obtain the following initial concentrations of NaOH (mM), substitute the following %B at Event Times 0, 8, 66.3, and 92 min in Table 1 above:
mM NaOH
%B
mM NaOH
%B
10
15
0.00
40
12.50
2.08
45
14.58
20
4.17
50
16.67
25
6.25
55
18.75
30
8.33
60
20.83
35
10.42
Tryptophan (Sigma Chemical Co)
Leucine (Sigma Chemical Co)
HEPES (Sigma Chemical Co)
Culture and Media
Dulbecco's modified Eagle's medium F12
(Sigma-Aldrich, Cat# D6421)
Technical Note 63
25 mM NaOH in 1 M Sodium acetate
To prepare 2 L of eluent, dissolve the contents of a
bottle containing 82 g of the AAA-Direct certified
anhydrous sodium acetate in ~800 mL purified water.
Adjust the total volume to 1.0 L with additional water.
Filter this solution through a 1-L 0.2-µm nylon filter unit
(see comments above). Repeat for a second bottle and
then gently combine into a 2-L plastic eluent bottle. Add
2.62 mL 50% NaOH to the 2.0 L sodium acetate solution, and immediately place it under 4–5 psi helium or
nitrogen to reduce carbonate contamination.
100 mM Acetic Acid
To prepare 2 L of eluent, add 11.5 mL of HPLC
Grade (99.7%) glacial acetic acid (17.5 M) to 1.5 L
purified filter-degassed water and then bring volume
to 2.0 L. Immediately place it under 4–5 psi helium or
nitrogen.
Keep the eluents blanketed under 5–8 psi (34–55
kPa) of inert gas (helium or nitrogen) at all times.
In-line degassing is necessary because amperometric
detection is sensitive to oxygen in the eluent.
STANDARD AND SAMPLE PREPARATION
Standards
Solid Leu and HEPES standards were maintained
desiccated under vacuum prior to use. They were dissolved in purified water to 10 g/L concentrations. These
were combined and further diluted with water to yield
the desired stock mixture concentrations. The solutions
were maintained frozen at -20 °C until needed. The
amino acid standard mix, SRM 2389, from NIST
(2.39–2.94 µM; except cystine, 1.16 µM) was diluted
250-fold with water to produce known concentrations of
each amino acid ranging from 9.5 to 11.75 µM (except
cystine, 4.6 µM). Tryptophan was also added to the
NIST amino acid standard mix during dilution.
Mammalian Cell Culture Medium
Dulbecco's modified Eagle's medium F12 (ingredients in references 6 and 7) was a sterile commercially
available (Sigma-Aldrich) ready-to-use liquid, diluted
1000-fold with water for analysis. Diluted sample was
analyzed directly.
SYSTEM PREPARATION AND SETUP
The preparation and setup of the AAA-Direct system
is described in the Product Manual for AAA-Direct
Amino Acid Analysis System.9 For optimal performance, it is important that the guidelines provided in this
manual be followed closely. Verify system performance
as described in the Product Manual. In an ICS-3000 using two separate channels and one AS autosampler, the
AS should be configured in Sequential Mode, using a
diverter valve, and each injection port volume accurately
calibrated prior to use10. The Chromeleon program file
for the ICS-3000 should be programmed to contain an
audit log command for pH, background, and backpressure at 0.00 min to assist in tracking system performance. The pH recorded by the reference electrode in
the electrochemical cell should remain ±0.5 pH units
from the theoretical pH for a given hydroxide eluent
applied to the beginning of the gradient program (e.g.,
60 mM NaOH should be pH 12.8, and 15 mM NaOH
should be pH 12.2). A deviation from this range is an indication of excessive reference electrode wear, and may
require its replacement (routinely every 6-12 months
for the ICS-3000 cell). Disposable electrodes should
be replaced after 7 days of continuous use. The background should remain within the range of 25–90 nC for
60 mM NaOH concentrations. Higher and lower values
may be an indication of disposable electrode malfunction, or contamination of the eluents, column, or both.
Variation in background is expected through the gradient program, as the NaOH concentration will change
during this program. The backpressure of the combined
new analytical and guard column set should be recorded
~1 h after it was first installed using 15–60 mM NaOH
at 0.25 mL/min. Typical backpressure for new column
sets range from 1960 to 2320 psi. The pressure should
range from ±500 psi thereafter. Excessive backpressure is an indication of blockage to either the plumbing
leading to the column, the frits of the guard column, or
column contamination. A contaminated column may be
cleaned following the instructions provided in the column manual. An increase in backpressure (~500 psi) is
expected through the gradient program, as the viscosity
will increase with increasing NaOH and sodium acetate
concentrations. Audit log values for pressure, pH, and
background may be easily trended using features available in Chromeleon. Maximum and minimum values
may be programmed to activate an alarm in Chromeleon
when values beyond these thresholds are detected.
Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry
EXTRACTION OF CHROMATOGRAMS
FROM THE 3-D Amperometry
DATA
The following procedure describes the steps required to extract
chromatograms needed to calculate
the numerators and denominators
used to generate the area ratios or the
height ratios. The process of optimizing intervals of time for current integration used to calculate numerators
or denominators is described in the
section “OPTIMIZATION OF CURRENT INTEGRATION RANGES
FOR RATIO CALCULATIONS”.
For the examples used in this section, the numerator and denominator
were based on the ranges of 240–480
ms, and 460–560 ms, respectively.
Figure 1.
These values may change as a result
of the optimization.
1. After chromatograms have
been collected, with 3-D amperometry data acquisition enabled, the
chromatogram is selected in the
browser and double clicked to present its report.
2. The Default report is displayed on the screen (Figure 1) with
either the ED_1 or the ED_1_Total
signal trace appearing in the report's
chromatographic window. Do not
zoom the peak of interest at this time,
and make sure both the x- and y-axes
are full scale. Single click the 3-D
amperometry button (Figure 1, label
A), which will then open the
3-D Amperometry window
(Figure 2). Note, if the 3-D amperometry display takes more than
10 seconds to open, then the chroFigure 2.
matographic trace in the report
window was probably not full scale.
To make sure chromatograms are full scale, double click
on the x- and y-axes prior to clicking the 3-D amperometry button. If the 3-D amperometry window was inadvertently opened without full scale, you must either wait
A
for the image to load before closing this window and
restarting with a full scale, or, alternatively, right-mouseclick to immediately end the graphics loading.
Technical Note 63
3. From the Menu Bar, pull
down View, and select Extract
– Chromatogram to file (single
click). This opens the Extract Amperometry Channel window (see
Figure 3).
4. Change the number in the
Begin Integration (ms) cell to the
desired number (e.g., "240"), and the
End Integration (ms) (e.g., "480"),
and then change the Channel name
cell to the desired signal file name.
We recommend naming your signal
file by the range used (i.e., “240 to
480 ms”) to easily identify the signal
range you are viewing at a later time.
5. Click the OK button to begin
extraction of the chromatogram with
the waveform integration period you
selected.
6. After about 5 s, the extracted chromatographic trace is
displayed in a new report, and the
report shows the channel name (the
signal file name you selected) in
the upper right corner (Figure 4).
The peak of interest may not be
integrated properly, and therefore
you may now zoom in on your peak
and modify the integration of this
peak. You may manually adjust the
begin and end of integration points
for this specific peak, or you may
adjust the Method by selecting View
– QNT-Editor from the Menu Bar,
in the same manner Method files
are modified in Chromeleon. When
two or more peaks nearly coelute,
and produce tails or fronts, integration should include the total area.
Also check that the retention time
correctly identifies the peak in the
Method file.
Figure 3.
Figure 4.
Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry
7. After the peak is properly
integrated and identified (Figure 5),
close this window by clicking the
lower X for this window at the top
right side of the screen (Figure 5,
label A). Chromeleon will prompt
you whether to save or not save the
changes you made to the peak integration (Figure 6). Click Yes.
8. After the extracted file has
been saved and the window closed,
the 3-D amperometry screen reappears. Repeat steps 3–7, but select
the second waveform integration
period to be used for the denominator
in the area response ratio calculation:
Change the number in the Begin
Integration (ms) cell to the desired
number (e.g., "460"), and the End
Figure 5.
Integration (ms) (e.g., "560"), and
then change the Channel Name cell
to the desired signal file name (i.e.,
“460 to 560 ms”). Check the second
extracted chromatogram for proper
peak integration.
9. After the second extracted
file has been saved and the window closed, the 3-D amperometry
screen reappears. Close the 3-D
amperometry window, and return
to the Browser.
10. Double click your selected
chromatogram in the browser,
which opens the Default Report
(Figure 1). From the Menu Bar,
pull down Workspace, and select
Load Report Definition. Under
the Local time base, open the folder named Dionex Templates, and
then the folder named Reports.
Figure 6.
Open the report named Peak Area
Ratios. This opens a report (Figure 7)
that calculates the peak area ratio for two signal files.
The signal files that the report uses for comparison is
defined in the two chromatogram properties, and the four
column properties used for the area and height ratio calculation of the report. These report properties must have
exactly matching names for the signal files that were
created above. To modify the report to accommodate
A
your file names, single left click the first chromatogram
window, then right click and select Chromatogram
Properties, then select the Channel tab, and then scroll
through the Fixed Channels and pick the one used
for the numerator in the ratio calculation. If you want
to modify other properties of the chromatogram, such
as narrowing the time scale (e.g., to look at a specific
Technical Note 63
peak or peaks) then adjust these properties now. Select
OK. Repeat this process for the second chromatogram
window, but selecting the Fixed Channel signal used
for the denominator. Next, link the columns to the new
signal file names by single clicking one of the cells in
the column labeled Numerator Area, then right click,
and select Report Column Properties. Scroll through
the Fixed Channels, and select the signal file name for
the numerator. Click OK. Repeat this linking to the next
column for Numerator Height. Also repeat this for the
next two columns for the denominator, but select the
signal file name being used for the denominator. Save
this report with a unique and descriptive name for your
future use.
11. When the report is configured correctly, it presents both the peak area and height ratios (Figure 7). When
a number of chromatograms have been extracted, the
toggle button (Figure 7, label A) can advance each chromatogram in the sequence to see their respective reports.
Figure 7.
OPTIMIZATION OF CURRENT INTEGRATION RANGES
FOR RESPONSE RATIO CALCULATIONS
To maximize the success of using area ratios or
height ratios to confirm peak identity or estimate impurity levels of coeluting peaks, the difference in the
numeric values calculated for the area ratio or height
ratio of two or more peaks should ideally be as large
as possible. Thus, the peak area or height used for the
numerator in one of the analytes needs to be as large as
possible and the denominator needs to be as small as
possible. At the same time, the other analyte must have
a peak area or height used for the numerator which is
as small as possible and the peak area or height used for
the denominator needs to be as large as possible. Also, to
maximize the validity of the area ratio and height ratio,
high precision for these values is desired. These conditions are obtained by varying the ranges used for current
integration in the numerator and denominator. The ranges used for 3-D amperometry extraction examples shown
in the “EXTRACTION OF CHROMATOGRAMS
FROM 3-D Amperometry DATA” section above were
240–480 ms for the denominator,
and 460–560 ms for the denominator. By incrementally increasing or
decreasing the limits of the numerator range, it may be possible to find
a combination that maximizes the
A
peak area or height differences in
the numerator between the two or
more compounds. The same strategy is applied to the denominator,
but with the intent of finding the
largest difference after reversing the
compounds subtracted from each
other, to ensure the area ratio and
height ratio will produce the largest
difference in value. For example,
compound A area is subtracted from
compound B area for the numerator
optimization, while compound B
area is subtracted from the compound A area for the denominator optimization. The
RESULTS AND DISCUSSION section below, with their
respective tables, shows an example of this optimization process comparing HEPES and Leu peak areas with
varying current integration ranges. Maximizing precision
is attained by selecting ranges that produce peak areas or
heights, for either the numerator or denominator, that are
Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry
significantly above background noise at the desired concentration, and produce peaks that have level baselines
making them easy to integrate. Besides selecting ranges,
precision is also affected by inconsistent peak integration settings and misidentified peaks. Many applications
apparently exhibit area ratio and height ratio values that
are independent of analyte concentration, as determined
by comparing these values at varying analyte concentrations (see RESULTS AND DISCUSSION section below).
Detection limit and linearity of detector response will also
impact these alternative current integration ranges and
therefore impact response ratios at these extremes. Selecting ranges that produce negative peaks should be avoided.
When sloping baselines cannot be avoided, ratios based
on peak area are generally better than peak height. Optimization often requires a balance of these variables. Compounds having similar chemical structures and functional
groups tend to exhibit similar area ratio and height ratio
values, and may not provide enough differentiation to be
statistically significant, and therefore 3-D Amperometry
may not be able to confirm peak identity or provide a
meaningful calibration slope required for estimation of
impurity levels in coeluting peaks.
RESULTS AND DISCUSSION
This section provides an example to demonstrate the
practical use of response ratios for peak identification
and their use for estimating concentrations of coeluting peaks. This example also demonstrates the response
ratio optimization process. Figure 8 shows that one of
the AAA-Direct methods (Table 1), separates all amino
acid and carbohydrate peaks expected to be present in
Dulbecco's Modified Eagle's: F-12 Ham Mixture.6,7 Of
particular interest in this medium was the seemingly
high concentration of Leu. The expected concentration
was 59 µg/mL, and the measured concentration was
990 µg/mL. The other amino acids were measured close
to their expected levels. The same results were obtained
with more dilute samples and when using other eluent
conditions described in Table 1, where selectivity for
some compounds is modified.6,7 The high concentration
of Leu was attributed to a manufacturing defect. By
comparing area ratios (or height ratios) for known
standards with unknown peaks, 3-D amperometry can
help confirm peak identity. The Dulbecco's media was
diluted (1000-fold) to produce a peak area equivalent to
a 10-µM injection of a Leu standard.
Column:
Eluent:
Temperature:
FlowRate:
Inj.Volume:
Detection:
Sample:
350
AminoPacPA10withGuard
AAA-Directgradient15/8
30°C
0.25mL/min
25µL
IPADwithdisposableAu
workingelectrode
Dulbecco'sModifiedEagle's
F-12Mixture(10-folddilution)
4
7
13
1
11.Proline
12.Isoleucine
13.Leucine
14.Methionine
15.Histidine
16.Phenylalanine
17.Glutamate
18.Aspartate
19.Cystine
20.Tyrosine
21.Tryptophan
19
15
11
10
nC
Peaks:
1. myo-Inositol
2. Arginine
3. Lysine
4. Glucose
5. Asparagine
6. Alanine
7. Threonine
8. Glycine
9. Valine
10. Serine
20
16
9 12 14
2
5
3
8
17 18
6
21
0
0
5
10
15
20
25
30 35
Minutes
40
45
50
55
60
65
22679
Figure 8. Separation of Dulbecco's Modified Eagle's F-12 Ham
Mixture (10-fold dilution, 25 µL) using the 15/8 gradient method
(Table 1).
Using the default non-optimized waveform integration
ranges of 220–460, and 470–560 ms for the numerator and denominator, the area ratio for the Leu standard
was 7703, and the area ratio of the questionable peak
in the medium was 5.63. This large difference in area
ratio values suggest that the peak is unlikely to be Leu.
Although the area ratio precision for replicate injections
of the media sample was high (1.2% RSD), the area ratio
precision for the Leu standard was not (81% RSD). The
Leu peak was barely detectable using the 470–560 ms
range (denominator), while a significant peak was observed for the Dulbecco's medium using the same range.
The integration of Leu peaks that were slightly above
baseline noise, and then used in the denominator of the
area ratio calculation, produced poor precision, making
statistical analysis impractical. Although this prevented
statistical evaluation, it was obvious from the area ratios
that the questionable peak was not Leu, and that further
current integration range optimization was needed. The
next step was to identify the unknown peak, and then optimize the current integration range settings used for the
area ratio (or height ratio) numerators and denominators.
Then, area ratio (or height ratio) calibration curves can
be generated to estimate the amount of Leu in the major
coeluting peak.
Technical Note 63
Table 2. Dulbecco's Modified Eagle’s: F-12 Ham
Mixture Ingredients with Unknown Retention Times
Vitamins
Other Components
Inorganic Salt
Biotin (D-)
HEPES
CaC2
Choline Chloride
Hypoxanthine
CuSO4
Folic Acid
Linoleic Acid
Fe(NO3)3
Inositol (i-, or myo-)
Putrescine
FeSO4
Nicotinamide
Sodium Pyruvate
KCl
Pantothenate (D-Ca-)
Thioctic Acid (DL-)
MgCl2
Pyridoxal HCl
Thymidine
MgSO4
Riboflavin
NaCl
Thiamine HCl
NaHCO3
Vitamin B12
Na2HPO4
NaH2PO4
ZnSO4
10
The composition of Dulbecco's media is documented,
and ingredient concentrations are known. After reviewing
the ingredient list provided by the manufacturer, the compounds listed in Table 2 have unknown retention times
on the AminoPac PA10.2,3,9 Most inorganic salts in Table
2, are not electrochemically active, but many vitamins
and other organic components are potentially active if
they contain an amine, hydroxyl, or a non-fully-oxidized
sulfur group. After injecting the most likely candidates,
HEPES was found to coelute with Leu, and its area ratio
determined to be 5.47 ± 0.11 (n = 4) using the waveform
integration ranges of 220–460, and 470–560 ms. This area
ratio was close to the value determined for the questionable peak in the Dulbecco's medium.
To estimate the amount of Leu present in the coeluting HEPES peak, higher precision of the area ratio for
Leu was required. To achieve this, optimization of the
waveform integration range was performed (see the
“OPTIMIZATION OF CURRENT INTEGRATION
RANGES FOR RESPONSE RATIO CALCULATIONS” section, above). Using previously collected 3-D
amperometry data for separate 10 µM Leu and HEPES
standard injections, the chromatograms were methodically extracted using varying start and end time values
for the current integration range (see below). The Leu
and HEPES peaks from these extracted chromatograms
were integrated and labeled using the Chromeleon
Quantitation (QNT) Editor. The results of this optimization are presented in Table 3.
Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry
Table 3. Results of Optimization of Current Integration Ranges for Response Ratio Calculations
Numerator
Period
(ms)
Denominator
Peak Area
(nC* min)
Area
Difference
Start
End
HEPES
Leu
(Leu-HEPES)
Period (ms)
Peak Area (nC*min)
Area
Difference
Start
End
HEPES
Leu
(HEPES-Leu)
10
460
6.55
6.36
-0.2
440
560
1.11
0.77
0.34
200
460
4.59
6.37
1.8
450
560
1.01
0.52
0.49
210
460
4.46
6.34
1.9
460
560
0.90
0.27
0.63
220
460
4.28
6.38
2.1
470
560
0.79
0.00
0.78
230
460
4.07
6.42
2.3
480
560
0.62
0.00
0.62
240
460
3.68
6.38
2.7
250
460
3.32
6.16
2.8
460
470
0.12
0.28
-0.16
260
460
3.05
5.98
2.9
460
520
0.60
0.30
0.30
270
460
2.76
5.72
3.0
460
530
0.67
0.30
0.37
450
460
0.10
0.25
0.1
460
540
0.74
0.29
0.45
460
550
0.81
0.28
0.53
240
440
3.51
5.87
2.4
460
560
0.90
0.27
0.63
240
450
3.55
6.08
2.5
460
590
0.89
0.28
0.61
240
460
3.68
6.38
2.7
240
470
3.82
6.60
2.8
240
480
4.01
6.77
2.8
240
490
4.11
6.71
2.6
240
500
4.18
6.69
2.5
Optimizing the Numerator
Holding the end point of current integration time
in the numerator at 460 ms, and varying the start time
from 10 to 450 ms, both Leu and HEPES peak areas
decreased with shortened duration, but the difference in
these areas (Leu Area – HEPES Area) showed a maximum between a current integration start time of 240–270
ms. Balancing the need for maximizing the difference in
peak area for Leu versus HEPES, and maximizing peak
area, the best numerator range start time was judged
to be 240 ms. Holding the integration start time in the
numerator at 240 ms, and varying the end time from 440
to 500 ms, both Leu and HEPES peak areas increased
with increased duration, but the difference in these areas
(Leu Area – HEPES Area) showed a maximum between
460–480 ms. Balancing the need for maximizing the difference in peak area for Leu versus HEPES, and maxi-
mizing peak area, the best numerator range higher value
was 480 ms. Therefore, the optimal numerator integration period is from 240 to 480 ms.
Optimizing the Denominator
Holding the end point of current integration period
used in the denominator at 560 ms, and varying the start
time of 440–480 ms, both Leu and HEPES peak areas
decreased with shortened duration, but the difference in
these areas (HEPES Area – Leu Area) showed a maximum between a current integration start time of 460–480
ms. Maximizing the difference in area ratio between Leu
and HEPES requires subtracting the extracted peak area
for HEPES from Leu used in the numerator, and then
subtracting the extracted peak area for Leu from HEPES
used in the denominator. This ensures the numerator
is the highest area value possible for HEPES, and the
denominator is the lowest area value possible for Leu in
order to make the difference in ratios between the two
Technical Note 63
11
Table 4. Effect of Variation of Duration of Current
Integration Period on Computed Area Ratios
Numerator
Period (ms)
Denominator
Period (ms)
Area Ratio
Area Ratio
Difference
Table 5. Averages of Area and Height Ratios
Peak Area Ratio*
Peak Height Ratio*
Sample
Mean
SD
RSD Mean
SD
RSD
Min
Max
Min
Max
HEPES
Leu
(Leu-HEPES)
Leucine
25.1
0.7
2.7%
22.1
0.7
2.9%
240
440
460
560
3.89
21.36
17.47
HEPES
4.4
0.1
2.7%
4.3
0.1
1.9%
240
450
460
560
3.94
22.11
18.17
Dulbecco's Medium** 4.9
0.2
3.8%
4.4
0.1
2.5%
240
460
460
560
4.08
23.19
19.11
n = 4 injections
240
470
460
560
4.24
24.01
19.77
240
480
460
560
4.44
24.62
20.18
* Ratio of peak area or height for waveform integration intervals of 240–480 ms / 460–560 ms for 10 µM HEPES and Leu.
240
490
460
560
4.56
24.40
19.85
240
500
460
560
4.64
24.34
19.70
compounds as high as possible. Unlike in the numerator,
the two shortest integration periods in the denominator
ranges (470–560 and 480–560 ms) produced peak area
values only slightly above baseline noise for Leu, and
were thus considered unacceptable. The period starting with 460 ms was acceptable, producing a measurable peak area. Holding the current integration start
time at 460 ms, and varying the end time of 470–590
ms, HEPES peak area increased with increased duration, while the Leu peak area decreased. The difference
between these areas (HEPES Area – Leu Area) was
maximized at 560 ms. Therefore, the optimal denominator current integration period was 460–560 ms.
Table 4 compares the effect of variation of duration
of current integration period on the computed area ratio
values for HEPES and Leu, and the difference in these
area ratio values. As discussed above, the greatest difference in area ratio between HEPES and Leu was attained
using the 240–480 ms and 460–560 ms ranges for the
numerator and denominator, respectively.
Peak Identification
Using the optimized numerator and denominator
ranges for Leu and HEPES, the area ratio and height ratio
values were calculated for both standards. Averages are
shown in Table 5 for 10 µM Leu and HEPES standards,
and a 1000-fold dilution of Dulbecco's medium. The area
ratios for HEPES and Leu are significantly different, and
as these values were determined from optimized current
integration ranges, it is now appropriate to perform a statistical evaluation. The area ratio for Leu was 25.1 ± 0.7,
and HEPES was 4.4 ± 0.1. The statistically different area
ratio for the two standards allows peak identification. The
12
** 1000-fold dilution
questionable peak found in Dulbecco's Medium showed
an area ratio value (4.9 ± 0.2 that was nearly identical
to the area ratio determined for HEPES. We believe the
slightly elevated area ratio for this peak compared to
the HEPES standard is due to the trace amounts of Leu
coeluting with HEPES in the sample. Similar results
were found using height ratio in this application, but peak
height ratios are often inaccurate. Imperfect coelutions
of peaks will cause peak broadening and peak height will
not reflect total concentration. For this reason, area ratio
is preferred over height ratio.
Estimation of Leucine Concentration in the Coeluting
HEPES Peak of a Cell Culture Media
Coelution of two or more compounds causes the
area ratio to shift in a proportional manner from one
area ratio extreme to the other. For this reason, it is
possible to create an area ratio calibration curve where
the measured area ratio may relate to the percent of
one compound relative to another in a coeluting peak.
The generation of an area ratio calibration curve is only
meaningful when a large enough difference exists in
the area ratios for these two compounds, the precision
or calibration sensitivity is sufficiently high, and area
ratio values are independent of concentration within the
required concentration range. In the example of Leu and
HEPES, the area ratios were optimized for difference
and precision, as described above. Next, it was necessary
to demonstrate that area ratio values for both compounds
were concentration independent. This was established
by injecting varying concentrations of each separate
standard through the range of concentrations intended
for analysis. The area ratio of each separate standard,
Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry
Table 6. Effect of Analyte Concentration on Area Ratio
HEPES Area Ratio
Leucine Area Ratio
Conc. (µM)
Mean
SD
n
RSD
Mean
SD
n
0.5
4.58
1.91
3
41.7%
1
4.67
0.32
2
6.9%
2
3.92
0.29
3
7.5%
3
4.06
0.02
3
0.6%
4
4.20
0.33
3
7.8%
5
4.34
0.31
3
7.2%
38.53
2.00
4
5.2%
7.5
RSD
4.15
0.04
3
1.0%
35.56
0.88
4
2.5%
10
4.12
0.03
3
0.6%
36.78
0.45
4
1.2%
12.5
4.08
0.04
3
0.9%
15
4.08
0.06
3
1.5%
33.79
0.85
4.20
36.17
Grand SD:
0.58
2.08
RSD:
13.7%
when plotted or tabulated against the injected concentration, should not show any significant trending. Table 6
compares HEPES and Leu area ratios at varying concentrations that are relevant to this example. Although slight
trending was observed for Leu, this was not considered
large enough to affect results significantly. Because
Leu and HEPES area ratios are concentration-independent, calibration curves are valid because the derived
slope and intercept assume each Leu and HEPES area
ratio remain constant at their potential concentrations
in an unknown sample. The Leu (and to a lesser extent
HEPES) area ratio values reported in Table 6 differ from
those in Table 5 because they were obtained from separate experiments where AminoPac PA10 columns and
disposable gold working electrodes had been changed.
Figure 9 shows the relationship of % Leu in HEPES
to the calculated area ratio for this mixture. As the
concentration of Leu relative to HEPES increases, the
area ratio value increases from the low value (e.g., 4.4)
observed for just HEPES (0% Leu), up to the high area
ratio value (e.g., 25.1) observed for just Leu (100%
Leu). The resulting curve may be used to calculate the
Leu concentration in unknown samples. The slope and
y-intercept for the first-degree polynomial regression
(r2 = 0.9827) of a curve ranging from 0–60% Leu in
HEPES were 0.123 and 4.089, respectively. When the
16
5.8% 9
8
7
6
AreaRatio
29
2.5%
Grand Mean:
N:
4
5
y=0.0993x+4.4201
R2=0.9949
4
3
2
1
0
0
5
10
15
20
PercentLeucineinHEPES
25
30
35
Figure 9. Percent leucine in HEPES area ratio calibration curve
using 240–480 / 460–560 ms range.
upper concentration was reduced to 33% Leu in HEPES
(closer to the concentrations of Leu in the HEPES peak
for the Dulbecco's medium sample), slope and y-intercept for the first-degree polynomial regression (r2 =
0.9949) were 0.099 and 4.420, respectively. The measured area ratio for HEPES without Leu was 4.433. The
calibration curve showed slight nonlinearity at higher
concentrations of Leu relative to HEPES. The reduction of the concentration range for calibration increased
accuracy, as reflected by the improved value of the
y-intercept (closer to the value obtained with the Leu
standard). The application of a second order (quadratic)
Technical Note 63
13
polynomial regression may be used to increase accuracy
for curvilinear relationships over a broader concentration
range. Using the first degree polynomial regression with
the range of 0–33% Leu in HEPES, the predominantly
HEPES peak in 1000-fold diluted Dulbecco's medium
was determined to contain 4.5% Leu, equivalent to 0.8
µM Leu. The measured Leu in the undiluted media was
therefore 750 µM, 167% of the manufactured target
level of 450 µM.
The lower limit of detection (LOD) for this method
is determined by multiplying the standard deviation of
the area ratio for HEPES at 0% Leu by 3, adding this to
the y-intercept, and using the slope and y-intercept to
solve for the % Leu. The LOD for this application was
2.4% Leu in HEPES, equivalent to 0.4 µM Leu in the
1000-fold diluted medium.
PRECAUTIONS AND RECOMMENDATIONS
Some factors affecting area ratio determinations and
impacting the ruggedness of this technique still remain
unknown at this time. The following are the known rules
for use of area ratio:
1. Do not calculate an area ratio for a specific
substance and then apply it to an experiment that uses a
different column, different working electrode, different
gradient program, different instrument or hardware, etc.
2. Area ratio characterization of peaks is not proof of
peak purity. Impurities having the same or similar area
ratio as the primary peak of interest are unlikely to show
a difference in area ratios when they coelute.
3. Differences in area ratios between impurities and
the primary peak of interest may be amplified by optimizing the current integration range, as described above.
4. When the mean area ratio for replicates of the primary peak of interest does not match the corresponding
standard tested under identical conditions, this suggests
the peak of interest is not pure and should be investigated further.
5. Collection of 3-D Amperometry data is a useful tool when needed, but when not required, should be
turned off in the program file to reduce data file size.
Computers used to support any 3-D Amperometry application should be equipped with a DVD+R drive or other
large storage media for data backup.
14
SUMMARY
3-D amperometry expands the analytical potential
of conventional amperometry. The continuous collection
of electrochemical data provides the opportunity to
evaluate optimal current integration after data collection.
Some practical applications available using this technique include identification of peaks and estimations of
the percent of a substance coeluting with another.
REFERENCES
1. LaCourse, W. R. Pulsed Electrochemical Detection
in High-Performance Liquid Chromatography. New
York: John Wiley & Son, 1997.
2. Determination of Amino Acids in Cell Cultures and
Fermentation Broths. Application Note 150, LPN
1538. Dionex Corporation, Sunnyvale, CA, July
2003.
3. Hanko, V. P.; Rohrer, J. S. Determination of Amino
Acids in Cell Culture and Fermentation Broth Media Using Anion-Exchange Chromatography with
Integrated Pulsed Amperometric Detection. Anal.
Biochem. 2004, 324, 29–38.
4. Determination of Carbohydrates, Alcohols, and Glycols in Fermentation Broths. Application Note 122,
LPN 1029. Dionex Corporation, Sunnyvale, CA,
October 1998.
5. Determination of Carbohydrates and Glycols in
Pharmaceuticals. Application Note 117, LPN 0957.
Dionex Corporation, Sunnyvale, CA, September
1997.
6. Hanko, V. P.; Heckenberg, A.; Rohrer, J. S. Determination of Amino Acids in Cell Culture and
Fermentation Broth Media Using Anion-Exchange
Chromatography with Integrated Pulsed Amperometric Detection. J. Biomol. Tech. 2004, 15, 315–
322.
7. An Improved Gradient Method for the AAA-Direct
Separation of Amino Acids and Carbohydrates in
Complex Sample Matrices. Application Update 152.
Dionex Corporation, Sunnyvale, CA.
8. Jandik, P.; Clarke, A. P.; Avdalovic, N.; Andersen,
D.; Cacia, J. Analyzing Mixtures of Amino Acids
and Carbohydrates Using Bi-Modal Integrated Amperometric Detection. J. Chromatogr. B. 1999, 732,
193–201.
Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry
9. Installation instructions and troubleshooting guide
for the AAA-Direct Amino Acid Analysis System.
Document No. 031481. Dionex Corporation, Sunnyvale, CA.
10. Making the Most of the AS Autosampler. Technical
Note 64, LPN 1756. Dionex Corporation, Sunnyvale,
CA.
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8. VWR Scientific Products, 3745 Bayshore Blvd.,
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vwrsp.com.
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Technical Note 63
15