Technical Note 63

Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry

INTRODUCTIONHigh Performance Anion-Exchange chroma-

tography with Pulsed Amperometric Detection (HPAE-PAD) or with Integrated Pulsed Amperometric Detec-tion (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 pre- or 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 complica-tions. 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 meth-od 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 volt-ages 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 there-fore 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, post-chromatographic 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 in-formation 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 stan-dard 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.

� Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry

In this technical note, we present detailed instruc-tions 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 manu-facturer. 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 impu-rity 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.

EQUIPMENTDionex 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 AutosamplerChromeleon Chromatography Management Software

CONDITIONSAAA-Direct ™ Method:

Columns: AminoPac® PA10 Analytical (P/N 55406) AminoPac PA10 Guard (P/N 55407)Flow Rates: 0.25 mL/minEluent: A: 10 mM NaOH B: 250 mM NaOH C: 25 mM NaOH/1 M Sodium acetate D: 100 mM Acetic acidInj. Volume: 25 µLTemperature: 30 °CDetection: Integrated pulsed amperometry, disposable

or conventional Au working electrodesGradientMethods: 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 concentra-tion, and it is held for 8 min before the start of the NaOH gradient.

Waveform Potential (V)Time(s) vs pH Gain Region Ramp Integration

0.00 +0.13 Off On Off 0.04 +0.13 Off On Off 0.05 +0.33 Off On Off 0.21 +0.33 On On On 0.22 +0.55 On On On 0.46 +0.55 On On On 0.47 +0.33 On On On 0.56 +0.33 Off On Off 0.57 -1.67 Off On Off 0.58 -1.67 Off On Off 0.59 +0.93 Off On Off 0.60 +0.13 Off On Off

TechnicalNote63 3

REAGENTS AND STANDARDSReagentsSodium hydroxide, 50% (w/w) (Fisher Scientific and

J. T. Baker)Deionized water, 18 MΩ-cm resistance or higherSodium acetate, anhydrous (AAA-Direct Certified,

Dionex Corporation, P/N 059326)Acetic acid, glacial (17.5 M; HPLC grade, 99.7%

minimum; J. T. Baker)StandardsAmino acid standard mix (NIST, Standard Reference

Material 2389)Tryptophan (Sigma Chemical Co)Leucine (Sigma Chemical Co)HEPES (Sigma Chemical Co)

Culture and MediaDulbecco's modified Eagle's medium F12

(Sigma-Aldrich, Cat# D6421)

PREPARATION OF SOLUTIONS AND REAGENTSSodium Hydroxide Eluents10mMand250mMSodiumhydroxide

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 spe-cies 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 car-bonate contamination.

0.0 5 100–%B * 0.0 0.0

8.0 5 100–%B * 0.0 0.0

14.0 8 66.7 33.3 0.0 0.0

17.0 5 66.7 33.3 0.0 0.0

24.0 8 1.0 89.0 10.0 0.0

27.0 5 1.0 89.0 10.0 0.0

30.0 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

40.0 5 30.0 30.0 40.0 0.0

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

*ToobtainthefollowinginitialconcentrationsofNaOH(mM), substitutethefollowing%BatEventTimes0,8,66.3,and92minin Table1above:

mM NaOH %B mM NaOH %B

10 0.00 40 12.50

15 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

Table 1. Modified AAA-Direct Gradient Methods

InitialNaOHEluentConcentration: XInitialIsocraticTime: 8minAAA-DirectMethodName: X/8

EventTime(min)

CurveType

%A10 mMNaOH

%B250 mM

NaOH

%C25 mMNaOH +

1 MSodiumAcetate

%D100 mMAceticAcid

� Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry

25mMNaOHin1MSodiumacetateTo 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 solu-tion, and immediately place it under 4–5 psi helium or nitrogen to reduce carbonate contamination.

100mMAceticAcidTo 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 PREPARATIONStandards

Solid Leu and HEPES standards were maintained desiccated under vacuum prior to use. They were dis-solved 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 MediumDulbecco's modified Eagle's medium F12 (ingre-

dients 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 SETUPThe preparation and setup of the AAA-Direct system

is described in the Product Manual for AAA-Direct Amino Acid Analysis System.9 For optimal perfor-mance, 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 us-ing 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 backpres-sure at 0.00 min to assist in tracking system perfor-mance. 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 in-dication 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 back-ground 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 malfunc-tion, or contamination of the eluents, column, or both. Variation in background is expected through the gradi-ent 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 backpres-sure 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 col-umn 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 avail-able in Chromeleon. Maximum and minimum values may be programmed to activate an alarm in Chromeleon when values beyond these thresholds are detected.

TechnicalNote63 �

Figure1.

Figure2.

EXTRACTION OF CHROMATOGRAMS FROM THE 3-D AMPEROMETRY DATA

The following procedure de-scribes 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 optimiz-ing intervals of time for current inte-gration used to calculate numerators or denominators is described in the section “OPTIMIZATION OF CUR-RENT INTEGRATION RANGES FOR RATIO CALCULATIONS”. For the examples used in this sec-tion, the numerator and denominator were based on the ranges of 240–480 ms, and 460–560 ms, respectively. These values may change as a result of the optimization.

1. After chromatograms have been collected, with 3-D amperom-etry data acquisition enabled, the chromatogram is selected in the browser and double clicked to pres-ent its report.

2. The Default report is dis-played 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 am-perometry display takes more than 10 seconds to open, then the chro-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 amperom-etry button. If the 3-D amperometry window was inad-vertently opened without full scale, you must either wait

for the image to load before closing this window and restarting with a full scale, or, alternatively, right-mouse-click to immediately end the graphics loading.

A

6 Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry

3. From the Menu Bar, pull down View, and select Extract – Chromatogram to file (single click). This opens the Extract Am-perometry 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 ex-tracted 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, integra-tion should include the total area. Also check that the retention time correctly identifies the peak in the Method file.

Figure3.

Figure4.

TechnicalNote63 �

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 inte-gration (Figure 6). Click Yes.

8. After the extracted file has been saved and the window closed, the 3-D amperometry screen reap-pears. 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 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 win-dow 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 fold-er named Dionex Templates, and then the folder named Reports. 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 cal-culation 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

Figure5.

Figure6.

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

� Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry

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 im-purity 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 condi-tions are obtained by varying the ranges used for current integration in the numerator and denominator. The rang-es 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 denomina-tor. By incrementally increasing or decreasing the limits of the numera-tor range, it may be possible to find a combination that maximizes the peak area or height differences in the numerator between the two or more compounds. The same strat-egy 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 com-

pound A area for the denominator optimization. The RESULTS AND DISCUSSION section below, with their respective tables, shows an example of this optimiza-tion 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 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 pres-ents 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 chro-matogram in the sequence to see their respective reports.

Figure7.

A

TechnicalNote63 �

significantly above background noise at the desired con-centration, and produce peaks that have level baselines making them easy to integrate. Besides selecting ranges, precision is also affected by inconsistent peak integra-tion 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 concentra-tions (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. Select-ing 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. Opti-mization often requires a balance of these variables. Com-pounds 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 DISCUSSIONThis section provides an example to demonstrate the

practical use of response ratios for peak identification and their use for estimating concentrations of coelut-ing 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.

Figure8. 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 numera-tor 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 ob-served 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 op-timize 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.

0 5 10 15 20 25

22679

30 35 40 45 50 55 60 650

350

nC

Minutes

11.Proline12.Isoleucine13.Leucine14.Methionine15.Histidine16.Phenylalanine17.Glutamate18.Aspartate19.Cystine20.Tyrosine21.Tryptophan

1

20

19

1817

16

15

14

13

12

11

10

9

8

7

65

4

3

2

21

Column: AminoPacPA10withGuardEluent: AAA-Directgradient15/8Temperature: 30°CFlowRate: 0.25mL/minInj.Volume: 25µLDetection: IPADwithdisposableAu workingelectrodeSample: Dulbecco'sModifiedEagle's F-12Mixture(10-folddilution)

Peaks:1. myo-Inositol2. Arginine3. Lysine4. Glucose5. Asparagine6. Alanine7. Threonine8. Glycine9. Valine10. Serine

10 Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry

The composition of Dulbecco's media is documented, and ingredient concentrations are known. After reviewing the ingredient list provided by the manufacturer, the com-pounds 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 question-able peak in the Dulbecco's medium.

To estimate the amount of Leu present in the coelut-ing 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 CALCULA-TIONS” section, above). Using previously collected 3-D amperometry data for separate 10 µM Leu and HEPES standard injections, the chromatograms were methodi-cally 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 opti-mization are presented in Table 3.

Vitamins Other Components Inorganic Salt

Biotin(D-) HEPES CaC2

CholineChloride Hypoxanthine CuSO4

FolicAcid LinoleicAcid Fe(NO3)3

Inositol(i-,ormyo-) Putrescine FeSO4

Nicotinamide SodiumPyruvate KCl

Pantothenate(D-Ca-) ThiocticAcid(DL-) MgCl2

PyridoxalHCl Thymidine MgSO4

Riboflavin NaCl

ThiamineHCl NaHCO3

VitaminB12 Na2HPO4

NaH2PO4

ZnSO4

Table 2. Dulbecco's Modified Eagle’s: F-12 Ham Mixture Ingredients with Unknown Retention Times

TechnicalNote63 11

Optimizing the NumeratorHolding 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 maxi-mum 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 dif-ference 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 integra-tion period is from 240 to 480 ms.

Optimizing the DenominatorHolding 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 maxi-mum 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

Numerator Denominator

Period Peak Area Area Period Peak Area Area (ms) (nC* min) Difference (ms) (nC*min) Difference

Start End HEPES Leu (Leu-HEPES) 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

Table 3. Results of Optimization of Current Integration Ranges for Response Ratio Calculations

1� Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry

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 start-ing with 460 ms was acceptable, producing a measur-able 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 dura-tion, while the Leu peak area decreased. The difference between these areas (HEPES Area – Leu Area) was maximized at 560 ms. Therefore, the optimal denomina-tor 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 differ-ence 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 IdentificationUsing 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 sta-tistical 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

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,

Numerator Denominator Area Area Ratio Period (ms) Period (ms) Ratio Difference

Min Max Min Max HEPES Leu (Leu-HEPES)

240 440 460 560 3.89 21.36 17.47

240 450 460 560 3.94 22.11 18.17

240 460 460 560 4.08 23.19 19.11

240 470 460 560 4.24 24.01 19.77

240 480 460 560 4.44 24.62 20.18

240 490 460 560 4.56 24.40 19.85

240 500 460 560 4.64 24.34 19.70

Table 4. Effect of Variation of Duration of Current Integration Period on Computed Area Ratios

Peak Area Ratio* Peak Height Ratio*

Sample Mean SD RSD Mean SD RSD

Leucine 25.1 0.7 2.7% 22.1 0.7 2.9%

HEPES 4.4 0.1 2.7% 4.3 0.1 1.9%

Dulbecco'sMedium** 4.9 0.2 3.8% 4.4 0.1 2.5%

n=4injections

*Ratioofpeakareaorheightforwaveformintegrationintervalsof 240–480ms/460–560msfor10µMHEPESandLeu.

**1000-folddilution

Table 5. Averages of Area and Height Ratios

TechnicalNote63 13

when plotted or tabulated against the injected concentra-tion, should not show any significant trending. Table 6 compares HEPES and Leu area ratios at varying concen-trations 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-indepen-dent, 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 sepa-rate 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

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-in-tercept for the first-degree polynomial regression (r2 = 0.9949) were 0.099 and 4.420, respectively. The mea-sured area ratio for HEPES without Leu was 4.433. The calibration curve showed slight nonlinearity at higher concentrations of Leu relative to HEPES. The reduc-tion 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)

Table 6. Effect of Analyte Concentration on Area Ratio

HEPES Area Ratio Leucine Area Ratio

Conc. (µM) Mean SD n RSD Mean SD n RSD

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 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 2.5%

Grand Mean: 4.20 36.17

Grand SD: 0.58 2.08

N: 29 16

RSD: 13.7% 5.8%

Figure9. Percent leucine in HEPES area ratio calibration curve using 240–480 / 460–560 ms range.

0

1

2

3

4

5

6

7

8

9

0 5 10 15 20 25 30 35PercentLeucineinHEPES

Area

Rat

io

y=0.0993x+4.4201R2=0.9949

1� Peak Identification and Estimation of Percent Purity using HPAE with 3-D Amperometry

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 RECOMMENDATIONSSome 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 opti-mizing the current integration range, as described above.

4. When the mean area ratio for replicates of the pri-mary 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 investi-gated further.

5. Collection of 3-D Amperometry data is a use-ful 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 appli-cation should be equipped with a DVD+R drive or other large storage media for data backup.

SUMMARY3-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 tech-nique include identification of peaks and estimations of the percent of a substance coeluting with another.

REFERENCES1. 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 Me-dia Using Anion-Exchange Chromatography with Integrated Pulsed Amperometric Detection. Anal. Biochem. �00�, 324, 29–38.

4. Determination of Carbohydrates, Alcohols, and Gly-cols 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. De-termination of Amino Acids in Cell Culture and Fermentation Broth Media Using Anion-Exchange Chromatography with Integrated Pulsed Ampero-metric Detection. J. Biomol. Tech. �00�, 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 Am-perometric Detection. J. Chromatogr. B. 1���, 732, 193–201.

TechnicalNote63 1�

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LPN1757PDF109/16©2016DionexCorporation

9. Installation instructions and troubleshooting guidefor the AAA-Direct Amino Acid Analysis System.Document No. 031481. Dionex Corporation, Sunny-vale, CA.

10. Making the Most of the AS Autosampler. TechnicalNote 64, LPN 1756. Dionex Corporation, Sunnyvale,CA.

LIST OF SUPPLIERS1. VWR Scientific Products, 3745 Bayshore Blvd., Bris-

bane, CA 94005, U.S.A. Tel: 800-932-5000, www.vwrsp.com.

2. Sigma Chemical Company, P.O. Box 14508, St.Louis, MO 63178-9916, U.S.A. Tel: 800-521-8956,www.sigma.sial.com.

Chromeleon, AminoPac, and BioLC are registered trademarks and AAA-Direct and AAA-Certified are trademarks of Dionex Corporation.

3. Nalge Nunc International, 75 Panorama Creek Drive,P.O. Box 20365, Rochester, NY 14602-0365, U.S.A.Tel: 716-586-8800, www.nalgenunc.com.

4. National Institute of Standards & Technology (NIST),100 Bureau Drive, Gaithersburg, MD 20899, U.S.A.Tel: 301-975-2000, www.nist.gov.

5. Fisher Scientific, 2000 Park Lane, Pittsburgh, PA15275-1126, U.S.A., Tel: 800-766-7000, www.fisher-sci.com.

6. Gast Manufacturing Corporation, 2300 SouthHighway M139, P.O. Box 97, Benton Harbor, MI49023-0097, U.S.A., Tel: 616-926-6171, www.gast-mfg.com.

7. Sigma Chemical Co., P.O. Box 952968, St. Louis, MO63195-2968, USA. Tel: 1-800-521-8956. www.sigma-aldrich.com.

8. VWR Scientific Products, 3745 Bayshore Blvd.,Brisbane, CA 94005, USA. Tel: 800-932-5000, www.vwrsp.com.