Calibration

In this Section, we will Discuss:

• Quantification theory.
• Calibration theory.
• Calibration menus and views.
• Creating a calibration table.
• Recalibration.
• Multi-level calibration.
• Modifying the calibration table.
• Calibration settings.

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What is Quantification?

Quantitative Analysis involves:

• Knowing the compound you are analyzing.
• Establishing a Method for analysis.
• Analyzing standards of known concentration.
• Analyze the sample containing an unknown concentration.
• Compare the response of the unknown sample response to the
response of the known concentration.

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Quantification Calculations

ChemStation offers the following Calculation Procedures:

• Percent
• Normalization
• External Standard (ESTD)
• ESTD%
• Internal Standard (ISTD)
• ISTD%

Calibration is the process of relating a particular peak height or

area quantity with a component concentration or quantity.

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Area Percent

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Percent: Uncalibrated Procedure

• Analysis of a sample containing

Area Counts Area %
unknown amounts of C-12, C14, C-
C12 280 26.2 16 and C-18.
C14 250 23.4
• Fatty Acid Methyl Esters (“Fame”)
by the Area % Method.
C16 220 20.6

C18 320 29.9

1070 100.1

280
Area % of C12 = X 100 = 26.2
280 + 250 + 220 + 320

AREA (pk)
Σ AREA (pk)
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External Standard Method

• Plot detector response versus quantity

– Response: area, height, current, etc.
– Quantity: concentration, mass, etc.

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Advantages of External Standard

• Simple to set-up.

• No (or few) transformation calculations.

• This method keeps you "closest" to your data.

– Same units as the standards you prepare and analyze.

• Each peak is computed independent of the other peaks in the

mixture.

• Only the peaks of interest need to be calibrated.

• Only peaks of interest need to be eluted and measured.

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Disadvantages of External Standard

• Does not correct for instrument or detector response drift.

– Common for many
systems.
• Does not correct for
variations in volume
analyzed.
– GC syringe malfunction.
– GC autosampler issues.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

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Example Calculation of External Standard
Method
Area Counts Area % RF
C12 250 20.2 25/250 = 0.100
C14 290 23.4 25/290 = 0.086
C16 330 20.6 25/330 = 0.076
C18 370 29.9 25/370 = 0.068
1240 100.0

Single point calibration of a standard sample containing 25 ng each of C-12, C-14, C-16,
and C-18 FAME.
The response factors are stored and used to determine the amount of each component in a
sample containing unknown amounts of C-12, C-14, C-16, and C-18.

RESPONSE FACTOR = AMOUNT / AREA

AMOUNT OF UNKNOWN = AREA(pk) X RF(pk)

Response factor takes into account that equal amounts do not yield equal detector
response.

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Normalization

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Normalization

RF (pk) X AREA (pk)

NORM % = X 100%
[RF (pk) X AREA (pk)]

Internal Standard (ISTD)

AREA (pk) X RF (pk) X ISTD conc.

AMOUNT =
AREA (ISTD) X RF (ISTD)

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Internal Standard Method

• Add fixed amount of internal standard to samples and standards.

– Usually added just before analysis

• Calculate Response Ratio.

– RRY = RA/RIS

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Calculating with Internal Standards

• Calculating Response Ratios.

• RRPeak 1 = RPeak 1/RIS

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Advantages of Internal Standard

• Corrects for volume variations.

– Responses for analyte and internal standard change by same
fraction.
• Corrects for long-term response changes.
– Between runs.
– Between days.

• Each peak is computed independent of the other peaks in the

mixture.

• Only the peaks of interest need to be calibrated.

• Only peaks of interest need to be eluted and measured.

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Disadvantages of Internal Standard

• Selecting appropriate internal standard compound.

– Response similar.
– Retention time OK.
• Adds complexity to sample.
– Another peak present.
• Additional solutions needed.
• Cost.
– Internal standard may be more expensive.

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Criteria for Choosing the Internal Standard

• Not present in the sample.

• Readily available.
• Chemically similar to sample.
• Same concentration range as sample.
• Does not react with sample.
• Elutes near components of interests.
• Isolated, clean peaks.
• Chromatographically stable.

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Internal Standard Calculation
Single point calibration of a standard
Calibration sample containing 25 ng each of
Area Counts RF Single point calibration of a standard
C12 250 0.100 sample containing 25 ng each of C-12,
C-14, C-15, C-16 and C-18 FAME
C14 290 0.086
where the C-15 FAME acts as the
C15 310 0.081
internal standard.
C16 330 0.076
Unknown Sample
C18 370 0.068
RF X Area ISTD CONC ISTD
Area Counts (ESTD) AREA ISTD X RF ISTD Result
C12 280 28.0 ng X 1.10 = 30.8 ng

C14 250 21.5 ng X 1.10 = 23.7 ng

C15 280 22.7 ng X 1.10 = 25.0

C16 220 16.7 ng X 1.10 = 18.4

C18 320 21.8 ng X 1.10 = 24.0 ng

ISTD Correction Factor: 25 / 22.7 = 1.10

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Single And Multi-Level Calibration

C A
C A
C A B
A B
B
B

1 0.5 1.5 1.5 1.0 2.0 2.0 1.5 3.0 3.0 2.0 4.0

LEVEL 1 LEVEL 2 LEVEL 3 LEVEL 4

SINGLE POINT FOR

COMPONENT A
AREA/HEIGHT

Level 4

AREA/HEIGHT
Level 3

Level 2

Level 1
Area
Response of
Unknown

CONCENTRATION CONCENTRATION
Calculated
Amount MULTI-LEVEL FOR
COMPONENT C

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Definitions

• Sensitivity
– Although often used to express a concept similar to limits of
detection, sensitivity is actually related to the slope of the
calibration curve.
• “High sensitivity” means a large change in response for a small
change in amount (or concentration) that produces the response.

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Calibration is Part of a Method

Instrument setup
Acquisition

Integration setup

Calibration setup

Report type setup

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Calibration Menus
Calibration View

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New Calibration Table

Enter calibration level.

Enter default amount for all peaks.

Select and Delete any peaks of non-interest

Notice curve

Enter compound name. Change amount (if necessary).

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Recalibrate: Calibration Table

Select level to
recalibrate.

Select
} recalibration
mode.

In re-calibration area is the variable resulting in a different Response factor. 24

Multi-Level Calibration

Specify level.

Specify default
amount for this
level.

Notice curve

Notice different areas and response factors at different levels.

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Expected Sample Range

• Standards must bracket the expected range.

• Calibration standards should not exceed expected range by more
than 2X.
– May cause calibration errors.

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 Bracketing Standards

• Standards must bracket expected sample range.

1000

100
<-Expected Sample Range->
10

1
1 10 100 1000

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Desired Accuracy

• Know your analytical needs.

• If desired accuracy is:
– Low
• Allow greater spacing of levels.
• Allow greater calibration range.
• Use fewer standards.
– High
• Reduce spacing.
• Restrict range.
• Use more standards.

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Add Extra Peaks to Calibration Table

Specify level.

Specify default
amount.

New peak(s) add to table in next run.

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Calibration Settings

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Advanced Calibration

Average recalibration Sequence recalibration and

for R.F. and/or R.T. Bracket recalibration printing. 31
Calibration Table Options

Selectable calibration table columns.

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Standard Calibration Tables

Select table view.

Overview
Table.

Compound
Details Table.

Peak Details
Table.
Identification
Details Table.
Manual Setup
Table.

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Calibration Control Sample Limit Table

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Calibration Retention Time Window

Enter user specified time window or calculated defaults (Def) will be used.
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Calibration Compound Groups

36
System Suitability: Edit Noise Ranges

37
Performance Limits Range Settings

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Lab Exercises

The Students will perform the following lab exercises:

• Lab 1: Creating a Single Level Calibration in an Off-Line

Session.

• Lab 2: Working with Calibration Settings.

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