Velocity-corrected area calculation: SCIEX PA 800 Plus
Empower Driver version 1.3 vs. 32 Karat™ Software
Firdous Farooqui1, Peter Holper1, Steve Questa1, John D. Walsh2, Handy Yowanto1
1SCIEX, Brea, CA
Waters Corporation, Milford, MA

2

Since the introduction of commercial capillary electrophoresis(CE) systems over 30 years ago, it has been important to notalways use conventional “chromatography thinking” when using
CE. This is especially true when processing data, as there aresomekey
differences
betweenelectrophoreticand
chromatographic data.
For instance, in most capillary electrophoresis separations, peakarea is not only a function of sample load, but also of an analyte’svelocity past the detection window. In this case, early migratingpeaks move past the detection window faster than later migratingpeaks. This creates a peak area bias, as any relative difference inan analyte’s migration velocity will lead to an error in peak area
determination and relative peak area percent. To help minimize
this bias, peak areas are normalized by migration velocity. The
resulting parameter is commonly referred to as corrected peakarea or velocity corrected area.
This technical note provides a comparison of velocity correctedarea calculations using 32 Karat™ and Empower software. For
both, standard processing methods without manual integration
were used to process each result. For 32 Karat™ software,
Caesar integration1 was turned off. For Empower software, both
ApexTrack and Traditional processing methods are used.

Key Features
 Calculation of velocity-corrected area (VCA)
 Correlation of VCA between 32 Karat™ software andEmpower software The average absolute and relative percent difference of %VCA between 32 KaratTM software and Empower software’s(Apex and Traditional) are comparable.

Methods
Instrument
All experiments were performed on a PA 800 Plus System(SCIEX, Brea, CA) equipped with a PDA detector. Data wascollected using the PA 800 Plus Empower™ Driver v1.3. A 50 µm
internal diameter bare-fused silica capillary (SCIEX, P/N 338451)
was installed to give 30.2 cm total length (effective length 20 cm).

Figure 1: The PA 800 Plus Pharmaceutical Analysis System.

The capillary temperature was maintained at 25°C in all
separations. The voltage was applied using reverse polarity.
The following methods were used with the SCIEX PA 800 Plus
Empower™ Driver v1.3:
 IgG_HR_Conditioning: conditions the capillary IgG_HR_Separation: performs IgG separation
 IgG_HR_Shutdown: cleans the capillary at the end of asequence for storage and turns off the UV lamp
Chemicals and materials:
The IgG Purity/ Heterogeneity Assay Kit (P/N A10663) was
obtained from SCIEX (Carlsbad, CA). 2-mercaptoethanol waspurchased from Millipore-Sigma (Burlington, MA).
Sample preparation
Prepare the IgG control standard (reduced)
1.

The IgG control standard (~ 1mL) was aliquoted into ~10 vialsof 95 µl each and stored at -20°C to avoid multiple freezethaw cycles.

2.

Thaw one of the 95 μL aliquots of the IgG control standard atroom temperature.

3.

Add 2 μL of the 10 kDa internal standard to the IgG tube.

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4.

Inside a fume hood, add 5 μL of 2-mercaptoethanol to the IgG
tube.

5.

Cap the tube and mix thoroughly.

6.

Using a centrifuge, spin the tube for 1 minute at 300xg.

7.

Seal the vial cap with Parafilm and then heat the vial at 70°Cfor 10 minutes.

8.

Put the vial in a room-temperature water bath to cool for atleast 3 minutes.

9.

Transfer 100 μL of the prepared sample to a micro vial, put
the micro vial in a universal vial, and then cap the universalvial.

The data was collected on a SCIEX PA 800 Plus System using
the SCIEX PA 800 Plus Empower Driver™ v1.3 and analyzed withthe Empower software. The same data set was imported into 32
Karat™ software, analyzed and the results of the two analyses
were compared.

Figure 3: Overlay of six analyses of the IgG control standard under
reducing conditions

Calculating velocity-corrected area
Velocity-corrected area is calculated using the following formula 3.
𝐴Results and discussion= 𝑣𝐴 =

Typical results for the reduced IgG control standard

Acorr = Velocity-corrected area

The IgG control standard used in this assay includes a controlled
percentage of non-glycosylated heavy chain, which provides botha resolution and a quantification benchmark2. Figure 2 shows one
analysis of the reduced IgG control standard. Figure 3 shows anoverlay of all six reduced IgG analyses used in the VCA analysis.

𝑣 = Velocity

𝐿 𝐴𝑡

A = Uncorrected peak area
Ld = Capillary effective length (in cm)
t = Migration time (in seconds)
Several important features can be seen from this equation:

Figure 2: Separation of the IgG control standard under reducing
conditions

1.

Velocities are measured terms. They are calculated by
dividing the capillary effective length (Ld) by the migration
time of the analyte peak.

2.

The capillary effective length, Ld, is important. The total lengthof the capillary (Lt) is not used.

3.

The migration time (t) is the time it takes an analyte to movefrom the inlet of the capillary to the detection window.

Every type of chromatography data system software has its own
method of integration and of calculating area. In the Empowersoftware, VCA can be calculated either by entering the formula ina custom field using a fixed Ld value or by creating another custom
field for capillary length. The resulting VCA value is the same
regardless of how the calculation is performed. Tables 1, 2 and 3
show the reproducibility of the analysis of the reduced IgGsamples using 32 Karat™ software and SCIEX PA 800 Plus
Empower™ Driver v1.3.

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Velocity-corrected area Custom Field
The formula for Velocity Corrected Area is entered as a Custom
Field in Empower software as shown in Figure 4.

Table 1: % Reproducibility of % VCA for reduced IgG calculatedusing 32 Karat™ software.
Average

Std. Dev.

%RSD% LC

32.0

0.079

0.25% NG

6.7

0.057

0.86% HC

61.3

0.065

0.11% NG of HC

9.8

0.078

0.80

Table 2: Reproducibility of % VCA for reduced IgG, calculated using
Empower software (ApexTrack).
Average

Std. Dev.

%RSD% LC

31.80.419

1.32% NG6.80.053

0.78% HC

61.4

0.380

0.62Figure 4: Velocity-corrected area formula entry on Empowersoftware% NG of HC

9.90.050

0.50

Once the formula is entered as a Custom Field, the capillary
length is entered in the processing method as shown in Figure 5.

Table 3: Reproducibility of % VCA for reduced IgG calculated using
Empower software (Traditional).

Figure 5: Capillary Effective Length entry in Empower ProcessingMethod

Empower integration algorithms
The Empower software provides two integration algorithms toprocess data: Traditional: Uses the slope threshold (first derivative) to detectpeaks.
 ApexTrack: Uses the peak apex as identified by the rate ofchange of the slope (second derivative) to detect peaks.
For VCA calculations using the Empower software, data wasanalyzed using both integration methods. Tables 1, 2 and 3 show
the VCA calculation of six analyses of reduced IgG using 32
Karat™ software and the Empower software ApexTrack andTraditional integration algorithms.

Average

Std. Dev.

%RSD% LC

32.0

0.297

0.93% NG6.80.034

0.50% HC

61.2

0.276

0.45

% NG of HC

10.00.039

0.39

Overall, the data is comparable between the two software
programs and the two integration algorithms.
A comparison was also performed by calculating the relative
percent difference between the average VCA generated with 32
Karat™ software and Empower software using either ApexTrackor Traditional integration algorithms (Table 4, 5).

Table 4: Comparison of average absolute and relative percent
difference of %VCA between 32 Karat™ software and Empowersoftware ApexTrack integration
Absolute Difference

Relative Percent
Difference

% LC

0.01

0.6% NG

0.03

3.1% HC

0.01

1.4% NG of HC

0.01

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Table 5: Comparison of average absolute and relative percent
difference of %VCA between 32 Karat™ software and Empowersoftware Traditional integration
Absolute Difference

Relative Percent
Difference

% LC

0.01

0.6% NG

0.02

2.5% HC

0.01

0.6% NG of HC

0.01

1.7The results show that data collected using the SCIEX PA 800 Plus
Empower™ Driver v1.3 can be successfully used to calculate
VCA.

Conclusion
Overall, the VCA values generated using the Empower software
are comparable to those generated using 32 Karat™ software.
The data reported here are for comparison purpose only.
Migration to the SCIEX PA 800 Plus Empower Driver can be donewith confidence since the results from Empower software arecomparable to those generated using the 32 Karat™ software.

References1.

Application of the Caesar Algorithm to Capillary
Electrophoresis. T-1841A.

2.

IgG Purity and Heterogeneity Assay Kit Application Guide
RUO-IDV-05-6935-B

3.

P/ACE MDQ User’s Guide A33115AB

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