Agilent ChemStation Software User Manual V2 PDF

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Summary of Content for Agilent ChemStation Software User Manual V2 PDF

Agilent ChemStation for UV-visible Spectroscopy

Understanding Your UV-visible Spectroscopy System

Understanding Your UV-visible Spectroscopy System

Notices Agilent Technologies, Inc. 2000, 2003-2008, 2011

No part of this manual may be reproduced in any form or by any means (including elec- tronic storage and retrieval or translation into a foreign language) without prior agree- ment and written consent from Agilent Technologies, Inc. as governed by United States and international copyright laws.

Manual Part Number G1115-90041

Edition 7/11

Printed in Australia

Agilent Technologies 679 Springvale Road Mulgrave, Australia

Research Use Only This product may be used as a component of an in vitro diagnostic system if the sys- tem is registered with the appropriate authorities and complies with the relevant regulations. Otherwise, it is intended only for general laboratory use.

Warranty The material contained in this docu- ment is provided as is, and is sub- ject to being changed, without notice, in future editions. Further, to the max- imum extent permitted by applicable law, Agilent disclaims all warranties, either express or implied, with regard to this manual and any information contained herein, including but not limited to the implied warranties of merchantability and fitness for a par- ticular purpose. Agilent shall not be liable for errors or for incidental or consequential damages in connec- tion with the furnishing, use, or per- formance of this document or of any information contained herein. Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms, the warranty terms in the sep- arate agreement shall control.

Technology Licenses The hardware and/or software described in this document are furnished under a license and may be used or copied only in accor- dance with the terms of such license.

Restricted Rights Legend If software is for use in the performance of a U.S. Government prime contract or subcon- tract, Software is delivered and licensed as Commercial computer software as defined in DFAR 252.227-7014 (June 1995), or as a commercial item as defined in FAR 2.101(a) or as Restricted computer soft- ware as defined in FAR 52.227-19 (June 1987) or any equivalent agency regulation or contract clause. Use, duplication or disclo- sure of Software is subject to Agilent Tech- nologies standard commercial license terms, and non-DOD Departments and Agencies of the U.S. Government will receive no greater than Restricted Rights as

defined in FAR 52.227-19(c)(1-2) (June 1987). U.S. Government users will receive no greater than Limited Rights as defined in FAR 52.227-14 (June 1987) or DFAR 252.227-7015 (b)(2) (November 1995), as applicable in any technical data.

Safety Notices

CAUTION

A CAUTION notice denotes a haz- ard. It calls attention to an operat- ing procedure, practice, or the like that, if not correctly performed or adhered to, could result in damage to the product or loss of important data. Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met.

WARNING

A WARNING notice denotes a hazard. It calls attention to an operating procedure, practice, or the like that, if not correctly per- formed or adhered to, could result in personal injury or death. Do not proceed beyond a WARNING notice until the indicated condi- tions are fully understood and met.

Microsoft is a U.S. registered trademark of Microsoft Corporation.

Software Revision This handbook is for B.04.xx revisions of the Agilent ChemStation software, where xx is a number from 00 through 99 and refers to minor revisions of the software that do not affect the technical accuracy of this hand- book.

In This Guide This manual describes the data processing operations of the Agilent 8453 spectrophotometer and the Agilent ChemStation for UV- visible spectroscopy. It describes the processes and transformations that the spectrophotometric data undergo between the time that they are acquired in the spectrophotometer and their output on the Agilent ChemStation display or printer. The manual is designed to enable you to follow good laboratory practice (GLP) guidelines; using the information in the manual, you will be able to understand the data processing calculations from beginning to end and perform the data evaluation manually.

1 Overview of Data Processing

This chapter gives an overall summary of how data are acquired and processed in the spectrophotometer, transferred to, and then processed and reported by the Agilent ChemStation.

2 Agilent ChemStation Registers and Views

This chapter explains the concepts of registers and views, which are the bases of data storage and display in the Agilent ChemStation.

3 Tasks

This chapter describes the parameters and operation of the four Agilent ChemStation tasks: Fixed Wavelengths, Spectrum/Peaks, Ratio/Equation, and Quantification.

4 Spectral Processing and Data Transformation

This chapter contains details of the how math functions are used to process raw spectral data, and how wavelength data are extracted from processed data for evaluation

5 Evaluation

This chapter explains the Agilent ChemStation evaluation procedures.

Understanding Your UV-visible Spectroscopy System 3

6 Reports

This chapter contains descriptions of format and contents of the reports that are available with the general purpose UV- visible software for the Agilent ChemStation

7 Interactive Math Functions

This chapter contains full explanations of the math functions, including their operation at limiting conditions

8 Configuration and Methods

This chapter explains details of configuration and describes the software structure and method operation

9 Verification

This chapter describes the process of system verification and describes each of the verification tests in detail. Verification is the process of evaluating your spectrophotometer to ensure that it complies with documented specifications.

4 Understanding Your UV-visible Spectroscopy System

Contents

1 Overview of Data Processing 9

Data Processing in the Agilent 8453 Spectrophotometer 10

Agilent 8453 11

Dark Current Correction 12 Stray Light Correction 12 Variance 12 Reference Spectrum 12 Absorbance Spectra 13 Wavelength Calibration 14 Invalid Data 14

Data Flow in the Agilent ChemStation 15

Data Flow for Samples 15 Data Flow for Standards 16

2 Agilent ChemStation Registers and Views 19

Registers 20

Structure and Contents of Registers 21 Data Structure of Spectra 23

Views 24

Relationship Between Views and Registers 24

3 Tasks 25

Fixed Wavelength Task 26

Spectrum/Peak 27

Ratio/Equation 28

Agilent ChemStation for UV-visible Spectroscopy Macro Programming Guide 5

Contents

Quantification 30

4 Spectral Processing and Data Transformation 33

Spectral Processing 34

Absorbance 34 Transmittance 34 Derivative 34

Data Transformation 35

Use Wavelengths 35 Background Correction 35

5 Evaluation 39

Evaluation using an Equation 40

Quantification 41

Beer-Lambert Law 41 Calibration 42 Calibration Results 48 Quantification of Unknown Samples 50

6 Reports 51

Method Report 53

Results Report 55

Automation Reports 56

7 Interactive Math Functions 57

Unitary Operations 58

Absorbance 58 Transmittance 59 Derivative 60 Scalar Add 63 Scalar Multiply 63

6 Agilent ChemStation for UV-visible Spectroscopy Macro Programming Guide

Contents

Binary Operations 64

Add 64 Subtract 66

8 Configuration and Methods 69

System Configuration 70

Configuring the Hardware 70

The Configuration File 72

Methods 73

The Structure of Methods 73 Method Files 74 On Using a Method 75

The ChemStation.ini File 77

The Directory Structure 78

File Formats 81

9 Verification 83

Configuration of Verification 84

Verification Tests 85

Wavelength Accuracy 85 Photometric Accuracy 88 Stray Light 90 Resolution 90 Noise 91 Baseline Flatness 92 Stability 93

Spectrophotometer Selftests 94

Flow Test 95

Index 97

Agilent ChemStation for UV-visible Spectroscopy Macro Programming Guide 7

Contents

8 Agilent ChemStation for UV-visible Spectroscopy Macro Programming Guide

Agilent ChemStation for UV-visible Spectroscopy Understanding Your UV-visible Spectroscopy System

1 Overview of Data Processing

Data Processing in the Agilent 8453 Spectrophotometer 10

Data Flow in the Agilent ChemStation 15

9Agilent Technologies

1 Overview of Data Processing Data Processing in the Agilent 8453 Spectrophotometer

Data Processing in the Agilent 8453 Spectrophotometer

The following sections give an outline of how the Agilent 8453 spectrophotometer evaluates and processes spectra. All of these processes are implemented in firmware within the instrument. Since raw data is generally considered to be the actual values delivered by the measurement device to the controller (that is, the absorbance values), only an overview of the internal data processing is given here. Details of the algorithms are not provided.

10 Understanding Your UV-visible Spectroscopy System

Overview of Data Processing 1 Agilent 8453

Agilent 8453

The optical system of the Agilent 8453 is shown in Figure 1. It uses two light sources, a deuterium and a tungsten lamp. The shutter consists of two parts; one serves simply as an optical on/off switch, the second is a filter used to reduce stray light in the UV range. The 1024- element diode- array is a series of silicon photodiodes which are sensitive to light. Each photodiode is connected to a capacitor which is recharged at a constant interval by the control electronics. When light falls on a photodiode its resistance changes proportional to the incident light and the capacitor is discharged. The amount of electricity required to recharge the capacitor at each recharge cycle is a measure of the light intensity.

The readout circuitry of the diode- array produces an analog signal which is fed to an analog- to- digital converter for conversion into the digital domain. The output from the A/D converter is a digital intensity spectrum expressed in counts.

Figure 1 Optical Path of the Agilent 8453 Spectrophotometer

Understanding Your UV-visible Spectroscopy System 11

1 Overview of Data Processing Agilent 8453

Dark Current Correction

When the shutter is closed and no light falls on the array there is still a low- level signal due to leakage from the capacitor. This is called the dark current. The dark current signal is continually measured and stored within the electronics. It is automatically subtracted from the signal obtained when the shutter is opened.

Stray Light Correction

In a standard measurement sequence, reference or sample intensity spectra are measured without and then with the stray light filter in the light beam. Without the filter the intensity spectrum over the whole wavelength range from 190- 1100 nm is measured. The stray light filter is a blocking filter with 50% blocking at 420 nm. With this filter in place any light measured below 400 nm is stray light. This stray light intensity is then subtracted from the first spectrum to give a stray light corrected spectrum.

Variance

The instruments read- out rate of the diode array is 100 msec. By means of the integration time, data can be acquired using multiples of the readout time. If an integration time greater than 100 msec is applied, multiple spectra, dependent upon the selected integration time, are acquired. An average spectrum is calculated and, if requested, variance data for each data are calculated in addition.

Reference Spectrum

The Agilent 8453 is a single beam spectrophotometer. To generate a sample spectrum two steps are required. First a reference intensity spectrum, I0, is measured. This is usually measured on the cuvette with solvent. This reference intensity spectrum is stored internally with the Agilent 8453.

12 Understanding Your UV-visible Spectroscopy System

Overview of Data Processing 1 Agilent 8453

Absorbance Spectra

For a sample, the intensity spectrum is measured and the absorbance spectrum is calculated (no stray light correction) using:

where

is the reference intensity measured with the blank

is the dark current measured immediately before the reference

is the intensity measured with the sample

is the dark current measured immediately before the sample.

This absorbance spectrum together with the variance of each data point (if requested) is then transmitted to the controller.

When stray light correction is activated, Is and I0 are also corrected in the range 190 400 nm by subtracting the measured stray light:

where

is the stray light intensity measured with the blank

is the stray light intensity measured with the sample

A Is Isd( ) I0 I0d( )( )log=

I0

I0d

Is

Isd

A Is Isd Is(stray)( ) I0 I0d I0(stray)( )( )log=

I0(stray)

Is(stray)

Understanding Your UV-visible Spectroscopy System 13

1 Overview of Data Processing Agilent 8453

Wavelength Calibration

The Agilent 8453 has a nominal sampling interval of 1 diode every 0.9 nm. Each spectrograph is calibrated in the factory using a series of emission lines from deuterium, zinc, argon and mercury lamps to obtain a calibration curve that relates each diode to a particular wavelength. The calibration parameters of this curve are stored within the ROM memory of each spectrograph. On site, this calibration can be checked by using the two emission lines from the deuterium lamp. With the aid of the calibration parameters, the firmware of the spectrophotometer determines absorbance values at 1 nm intervals and these values are then sent to the controller.

Invalid Data

The Agilent 8453 will invalidate a measured spectrum under certain circumstances. This means that the measured spectrum is considered to be so unreliable that it is not usable. In the spectrum sent to the controller the absorbance values at wavelength which are invalidated are set to a special value which is recognized by the controller software and these values are not displayed. Invalid data are produced when the sample is much more transparent then the reference data of the previously acquired blank. This causes an A/D converter overflow.

14 Understanding Your UV-visible Spectroscopy System

Overview of Data Processing 1 Data Flow in the Agilent ChemStation

Data Flow in the Agilent ChemStation

The data flow in the Agilent ChemStation software can be considered as two separate processes for samples and standards. The data flows are linked by the calibration data that is produced during the processing of standards and used in the evaluation of samples.

Data Flow for Samples

Figure 2 shows the flow of sample data through the Agilent ChemStation registers from the transfer of the sample absorbance values and statistics from the spectrophotometer to the Agilent ChemStation to the production of a printed report. For qualitative evaluations with no quantification, the calibration data is not included in the evaluation.

Figure 2 Data Flow for Samples

Understanding Your UV-visible Spectroscopy System 15

1 Overview of Data Processing Data Flow in the Agilent ChemStation

Data Flow for Standards

Data flow during the calibration process, which is concerned only with standard samples, is shown in Figure 3.

Figure 3 Data Flow for Standards

16 Understanding Your UV-visible Spectroscopy System

Overview of Data Processing 1 Data Flow in the Agilent ChemStation

The calibration coefficients that are calculated during the calibration process are stored with the data analysis parameters in the Data Analysis Parameters register as part of the current method.

Once a calibration has been performed, and the calibration coefficients have been added to the Data Analysis Parameters register, the quantitative evaluation procedure uses the stored calibration data to perform a quantitative analysis on all subsequent samples that are measured using the current method. It is important to note that no data are destroyed during any of the data processing operations.

Understanding Your UV-visible Spectroscopy System 17

1 Overview of Data Processing Data Flow in the Agilent ChemStation

18 Understanding Your UV-visible Spectroscopy System

Agilent ChemStation for UV-visible Spectroscopy Understanding Your UV-visible Spectroscopy System

2 Agilent ChemStation Registers and Views

Registers 20

Views 24

19Agilent Technologies

2 Agilent ChemStation Registers and Views Registers

Registers

A register is an area of computer memory where data are stored. The General Scanning software sets up several registers for different types of data; the most important registers are shown in Table 1.

Table 1 Agilent ChemStation Registers and Their Contents

Register Contents

Configuration System configuration data (see Chapter 8, Configuration and Methods)

Sample System Parameters

Sampling system parameters to be used for measurements

Spectrophotometer Parameters

Spectrophotometer measurement parameter

Blank Spectrum Last acquired blank spectrum

Raw Data (Sample Spectra)

Unprocessed data from sample measurements (usually absorbance data)

Raw Data (Standard Spectra)

Unprocessed data from standard sample measurements (usually absorbance data)

Math Result Results of interactive math processing

Method Description Definition of the method (see Chapter 8, Configuration and Methods)

Data Analysis Parameters Definitions and parameters of all data processing steps

Processed Sample Spectra

Spectral data from samples after spectral processing

Processed Standard Spectra

Spectral data from standards after spectral processing

20 Understanding Your UV-visible Spectroscopy System

Agilent ChemStation Registers and Views 2 Registers

The registers which are specific to the standard samples and calibration data are used in the Quantification task only. During Quantification, the Data Analysis Parameters register includes the calibration coefficients generated during calibration process.

In addition, registers are set up for instrument parameters, method and automation parameters and analysis results. Temporary registers are also set up during data processing and during the execution of tasks.

Structure and Contents of Registers

All registers have the same basic structure, which is similar to a database or card- index system. The fundamental unit of the register is the object, which is equivalent to a record in a database or a single card in a card index system. For example, a measured spectrum is an object in the raw data register. Each object has three parts:

the object header, which contains static information about the object,

a group of tables, which contain dynamic information about the object,

the data block, which contains the objects data.

A graphical representation of the structure of a register is shown in Figure 4 on page 22.

Wavelength Results Function results from the specified wavelengths of the sample spectra

Wavelength Results Standards

Function results from the specified wavelengths of the standard spectra

Evaluation Results Evaluation or analysis results

Evaluation Results Standards

Evaluation results that are used for calibration diagnostics

Table 1 Agilent ChemStation Registers and Their Contents (continued)

Register Contents

Understanding Your UV-visible Spectroscopy System 21

2 Agilent ChemStation Registers and Views Registers

Figure 4 Register Structure

22 Understanding Your UV-visible Spectroscopy System

Agilent ChemStation Registers and Views 2 Registers

Data Structure of Spectra

The data structure of spectra is the same in the Samples register and the Math Result register:

Header block, containing some or all of the following information:

Information about the sample:

the sample name,

solvent information (if available),

any available comment,

the operator name,

the data type (absorbance).

Information about the instrument on which the sample was run:

the identification of the type and firmware revision number,

the instrument serial number (Agilent 8453).

Information about the acquisition:

the date and time of acquisition,

the integration time,

the resolution,

cell path length and units,

cell temperature (if available).

The Analytes Table, containing the information about the analytes. If no analyte information is available, this table is empty.

The Data Block, containing the spectral information in a matrix of three rows:

the wavelength in ascending order,

the absorbance at that wavelength,

the standard deviation of the measurement, calculated from the variance data transferred from the spectrophotometer.

Understanding Your UV-visible Spectroscopy System 23

2 Agilent ChemStation Registers and Views Views

Views

Views are perspectives on the contents of registers. Each view displays the contents of the relevant registers in one or more windows; each window of the view displays a portion of the registers contents.

Relationship Between Views and Registers

The Samples view and Math view display the contents of the Processed Samples register and Math Result register respectively:

The Sample Spectra window and Math Result window are graphical representations of the Data Blocks from the respective registers.

The Sample/Result table of the Samples view contains selected information from the Header block, together with the evaluation results The Samples Table, accessible from the Sample/Result table, contains additional information from the Header block.

The Standards view displays the contents of the Processed Standard Spectra register.

24 Understanding Your UV-visible Spectroscopy System

Agilent ChemStation for UV-visible Spectroscopy Understanding Your UV-visible Spectroscopy System

3 Tasks

Fixed Wavelength Task 26

Spectrum/Peak 27

Ratio/Equation 28

Quantification 30

25Agilent Technologies

3 Tasks Fixed Wavelength Task

Fixed Wavelength Task

For details of the calculations used in this task, see Chapter 5, Evaluation.

The Fixed Wavelength task allows the calculation of results from up to six wavelengths with or without background correction. The Fixed Wavelength parameters are shown in Table 2:

Table 2 Parameters for the Fixed Wavelength Task

Parameter Data Flow Range of Values

Use Wavelength(s) Sets the Used Wavelengths (see Use Wavelengths on page 35) to the selected values.

Up to six single wavelengths

Background Correction Sets the Background Correction (see Background Correction on page 35) to the selected type.

none single reference wavelength subtract average over a range three-point drop line

Data Type Sets the Spectral Processing (see Spectral Processing on page 34) to the selected type.

Transmittance Absorbance Derivative 1 Derivative 2 Derivative 3 Derivative 4

Display Spectrum Sets the upper and lower wavelength limits for spectral display.

Instrument Settings

26 Understanding Your UV-visible Spectroscopy System

Tasks 3 Spectrum/Peak

Spectrum/Peak

For details of the calculations used in this task, see Chapter 5, Evaluation.

The Spectrum/Peaks task determines the maxima and minima of the data points (y- values) in the defined wavelength range of the spectrum The last acquired spectrum is displayed annotated with the specified number of peaks and valleys, and the Sample Result table shows the wavelengths and absorbance values of the peaks and valleys. The All Peaks/Valleys button displays a list if the wavelengths and absorbencies of all peaks and valleys.

The original spectrum is first derivatized (see Derivative on page 60) using a filter length of 5 and a polynomial degree of 3. The derivatized y- values are examined for transitions (change of sign); each transitional y- value is compared with its neighboring values, and if the neighboring values are more extreme, the transitional y- value is compared with the previous transitional y- value. If the difference of both values is greater than or equal to the sensitivity threshold, then the previous transitional point is stored as an extremum; if the difference is less than the sensitivity threshold, then neither point is stored.

The Spectrum/Peaks parameters are shown in Table 3:

Table 3 Parameters for the Peak / Valley Find Task

Parameter Data Flow Range of Values

Data Type Sets the Spectral Processing (see Spectral Processing on page 34) to the selected type.

Transmittance Absorbance Derivative 1 Derivative 2 Derivative 3 Derivative 4

Display Spectrum Sets the upper and lower wavelength limits for spectral display

Instrument Settings

Number of Peaks Sets the number of peaks reported. 0 to 19

Number of Valleys Sets the number of valleys reported. 0 to 19

Understanding Your UV-visible Spectroscopy System 27

3 Tasks Ratio/Equation

Ratio/Equation

For details of the calculations used in this task, see Chapter 5, Evaluation.

The Ratio/Equation task calculates a result based upon a specified equation. The equation can contain absorbencies from up to six wavelengths plus sample weight and volume information. The following mathematical operators are available for use in an equation:

The results of the equation are corrected for path length and dilution factor automatically.

The parameters for the Ratio/Equation task are shown in Table 4:

+ adds

- subtracts

/ divides

* multiplies

ABS(exp) returns the absolute value of the expression

Ceil(exp) rounds the expression up to the nearest integer

EXP(exp) returns

FLOOR(exp) rounds the expression down to the nearest integer

LN(exp) returns the natural logarithm (base e) of the expression

LOG(exp) returns the logarithm (base 10) of the expression

SQR(exp) returns the square of the expression

SQRT(exp) returns the square root of the expression

e exp( )

28 Understanding Your UV-visible Spectroscopy System

Tasks 3 Ratio/Equation

Table 4 Parameters for the Ratio/Equation Task

Parameter Data Flow Range of Values

Use Wavelength(s) Sets the Used Wavelengths (see Use Wavelengths on page 35) to the selected values.

Up to six single wavelengths

Data Type Sets the Spectral Processing (see Spectral Processing on page 34) to the selected type.

Transmittance Absorbance Derivative 1 Derivative 2 Derivative 3 Derivative 4

Equation Name Sets the equation name. Alphanumeric text

Equation Sets the equation parameters. Alphanumeric text

Unit Sets the units for the result. Alphanumeric text

Use sample weight and volume

Switches on the Weight and Volume entry fields in the Sample Information dialog box.

Checked (yes) or unchecked (no)

Prompt for sample information

Switches on the display of the Sample Information dialog box after sample measurement.

Checked (yes) or unchecked (no)

Display Spectrum Sets the upper and lower wavelength limits for spectral display

Instrument Settings

Understanding Your UV-visible Spectroscopy System 29

3 Tasks Quantification

Quantification

For details of the calculations used in this task, see Chapter 5, Evaluation.

The Quantification task calculates calibration coefficients from the measured data of a set of standards, then uses the calibration coefficients to determine the concentration of unknown samples. The equations used in the calculation of the calibration coefficients and the concentrations of unknown samples are given in Chapter 5, Evaluation. The parameters for the Quantification task are shown in Table 5:

Table 5 Parameters for the Quantification Task

Parameter Data Flow Range of Values

Use Wavelength Sets the Used Wavelengths (see Data Transformation on page 35) to the selected values.

Single wavelength value

Background Correction Sets the Background Correction (see Background Correction on page 35) to the selected type.

none single reference wavelength subtract average over a range three-point drop line

Analyte name Sets the analyte name. Alphanumeric text

Calibration Curve Type Sets the calibration curve type (see Calibration Curve Types on page 42.)

linear (Beers law) linear with offset quadratic quadratic with offset

Concentration unit Sets the units of concentration; when the Concentration option is chosen, switches on the Concentration entry field in the Sample Information dialog box.

alphanumeric text

Weight & Volume unit Sets the units of weight and volume; when the Weight & Volume option is chosen, switches on the Weight and Volume entry fields in the Sample Information dialog box.

alphanumeric text

30 Understanding Your UV-visible Spectroscopy System

Tasks 3 Quantification

Prompt for standard information

Switches on the display of the Standard Information dialog box after sample measurement.

Checked (yes) or unchecked (no)

Prompt for sample information

Switches on the display of the Sample Information dialog box after sample measurement.

Checked (yes) or unchecked (no)

Data Type Sets the Spectral Processing (see Spectral Processing on page 34) to the selected type.

Transmittance Absorbance Derivative 1 Derivative 2 Derivative 3 Derivative 4

Display Spectrum Sets the upper and lower wavelength limits for spectral display

Instrument Settings

Table 5 Parameters for the Quantification Task (continued)

Parameter Data Flow Range of Values

Understanding Your UV-visible Spectroscopy System 31

3 Tasks Quantification

32 Understanding Your UV-visible Spectroscopy System

Agilent ChemStation for UV-visible Spectroscopy Understanding Your UV-visible Spectroscopy System

4 Spectral Processing and Data Transformation

Spectral Processing 34

Data Transformation 35

33Agilent Technologies

4 Spectral Processing and Data Transformation Spectral Processing

Spectral Processing

Absorbance

Absorbance is the default data type of spectral storage in the UV- visible ChemStation.

Absorbance spectra are calculated using the Absorbance function; for a complete description of the mathematical processes involved in calculating absorbance spectra, see Absorbance on page 58.

Transmittance

Transmittance spectra are calculated using the Transmittance function; for a complete description of the mathematical processes involved in calculating transmittance spectra, see Transmittance on page 59.

Derivative

First, second, third and fourth order derivative spectra can be calculated. The Derivative functions calculate the derivative of the data points (y- values) in the spectrum to the specified derivative order using a Savitsky- Golay algorithm with a filter length of 5 and a polynomial degree of 3. For a complete description of the Derivative function, see Derivative on page 60.

34 Understanding Your UV-visible Spectroscopy System

Spectral Processing and Data Transformation 4 Data Transformation

Data Transformation

Use Wavelengths

The Use Wavelengths function selects a sub- set of data from the full spectrum. The sub- set may be the processed spectral intensity value at a single wavelength, as in the Quantification task, or the processed spectral intensity values at up to six wavelengths as in the Fixed Wavelengths and Ratio/Equation tasks. The extracted value(s) may then be subjected to further modification by background correction to produce the final result.

Background Correction

Background correction subtracts a calculated background value from the extracted spectral intensity value(s). There are three methods of calculating the background values:

single reference wavelength

subtract average over a range

three- point drop line, used when two wavelengths are set

Single Reference Wavelength

The single reference wavelength method subtracts the spectral intensity value at a specified wavelength, as in Figure 5 on page 36. The reference wavelength is usually selected at a point on the baseline beyond the sample absorbance.

Understanding Your UV-visible Spectroscopy System 35

4 Spectral Processing and Data Transformation Data Transformation

Subtraction is carried out using Equation 1:

(1)

where

f is the function result at wavelength

A is the absorbance at wavelength

AR is the absorbance at reference wavelength

The variances (if available) are treated according to Equation 2:

(2)

where

var(f) is the variance of the function result at wavelength

var(A) is the variance of the absorbance at wavelength

var(AR) is the variance of the absorbance at reference wavelength

Figure 5 Background Correction to a Single Reference Wavelength

f A AR =

var f( ) var A( ) var AR ( )+=

36 Understanding Your UV-visible Spectroscopy System

Spectral Processing and Data Transformation 4 Data Transformation

Subtract Average Over Range

The subtract average over range method uses the same calculation as for a single reference wavelength (see Single Reference Wavelength on page 35), but replaces the spectral intensity value at the single wavelength with the average intensity value over a specified wavelength range.

where the terms are the same as for a single reference wavelength (see Background Correction on page 35).

Three-Point Drop Line

For a three- point drop- line background correction, the spectral intensity and variance values from two reference wavelengths are taken, giving ,

, and . In this case, the reference wavelengths define a

straight line (as in Figure 6 on page 38) which is used to calculate and using Equation 3 and Equation 4.

(3)

(4)

where the terms are the same as for a single reference wavelength (see Background Correction on page 35). In Figure 6 on page 38, the upper trace shows the spectrum before background correction, with the three- point drop line superimposed. The lower trace shows the spectrum after background correction.

f A= AR1

A+ R2

AR3 ARn+ + +( )

n -------------------------------------------------------------------

AR1

AR2 var AR1

( ) var AR2 ( )

AR var AR

( )

AR

1 R2

R1

--------------------- R2 ( )AR1

R1 ( )AR2

+{ }=

var AR ( ) 1

R2 R1

( )2 ----------------------------- R2

( )2var AR1 ( ) R1

( )2var AR2 ( )+

=

Understanding Your UV-visible Spectroscopy System 37

4 Spectral Processing and Data Transformation Data Transformation

Figure 6 Three-point Drop Line

38 Understanding Your UV-visible Spectroscopy System

Agilent ChemStation for UV-visible Spectroscopy Understanding Your UV-visible Spectroscopy System

5 Evaluation

Evaluation using an Equation 40

Quantification 41

This chapter describes the use of equations, and introduces quantification and explains the concepts that are used in the Quantification task of the software. It also describes the mathematical and statistical calculations that are used to produce a calibration curve for single component analysis and how the quantitative results are calculated using the calibration data.

39Agilent Technologies

5 Evaluation Evaluation using an Equation

Evaluation using an Equation

When an equation evaluation is specified, the analysis results are the results of the equation specified in the Equation Parameter dialog box. In the definition of the equation, you can use any result generated by the Use Wavelength(s) and stored in the Wavelength Results register and any of the other predefined variables: Dilution factor, Weight and Volume.

By default, the results are normalized to a 1 cm path length by dividing the function result by the value of the Path Length parameter in the Sample Spectra table:

(5)

where

fcorr is the function result after path length correction

f is the uncorrected function result

l is the path length

fcorr f l -=

40 Understanding Your UV-visible Spectroscopy System

Evaluation 5 Quantification

Quantification

The Quantification task of the Agilent ChemStation software includes facilities for:

measurement of calibration standards,

calibration of a single analyte using the calibration standards,

measurement of unknown samples, and

quantification of the analyte in the unknown samples.

Beer-Lambert Law

The Beer- Lambert law states that the absorbance of a solute is directly proportional to its concentration:

(6)

In Equation 6:

A is absorbance

E is molar absorptivity or molar extinction coefficient (l.mol- 1.cm- 1)

c is analyte concentration (mol.l- 1)

d is cell path length (cm)

To calculate the concentration of an unknown compound, the above equation can be solved for a given function result, f:

(7)

The reciprocal of the product of molar extinction coefficient and cell path length is often called the calibration coefficient, k.

(8)

A Ecd=

c f Ed ------=

c kf=

Understanding Your UV-visible Spectroscopy System 41

5 Evaluation Quantification

Calibration

Calibration is the correlation of the function result with known concentrations of sample. The basic assumption is that the variance in the measured data is less than the variance in the standard concentrations. As a result of this correlation a calibration curve can be fitted to the data points and calibration coefficients can be calculated. The concentrations of unknown samples can then be calculated from the calibration coefficients and the function result of the samples.

Calibration Curve Types

Real data may deviate slightly from the ideal linear relationship described in Beer- Lambert Law on page 41. The relationship between the function result and concentration may be described more accurately by adding an offset (a non- zero intercept) or a quadratic term (or both) to the equation.

The ChemStation provides four different calibration curve types, shown in Table 6. These calibration curve graphs are shown in the more traditional way with the concentration on the x- axes and the function results on the y- axes.

The Agilent ChemStation provides four different calibration curve types, shown in Table 6.

42 Understanding Your UV-visible Spectroscopy System

Evaluation 5 Quantification

c is the concentration

f is the function result

k0, k1, k2 are calibration coefficients

Number of Standards

There is no fixed limit to the number of calibration standards that can be incorporated into a calibration; the more points that can be placed on the curve, the more accurately the curve can be characterized. However, each of the calibration curve types requires a minimum number of standards of

Table 6 Calibration Curve Types for Single Component Analysis

Curve Type Curve Equation

I Linear without zero offset

II Linear with zero offset

III Quadratic without zero offset

IV Quadratic with zero offset

c k1f=

c k0 k1f+=

c k1f k2f 2

+=

c k0 k1f k2f 2

+ +=

Understanding Your UV-visible Spectroscopy System 43

5 Evaluation Quantification

different concentrations (see Table 7).

Calibration Standards

If the absorbance of the analyte at the wavelength(s) you select strictly obeys Beers Law, then one standard is sufficient to characterize the calibration curve (linear without zero offset). However, the use of two standards at different concentrations will confirm adherence to Beers Law, or show up any irregularities. Two standards are necessary to characterize a linear calibration curve with zero offset. Addition of a third standard at a different concentration is sufficient to identify and characterize a non- linear calibration curve.

Table 7 Minimum Number of Standards for Calibration Curves

Calibration Curve Type Minimum Number of Standards

Linear, no zero offset (Beers law) 1

Linear with zero offset 2

Quadratic, no zero offset 2

Quadratic with zero offset 3

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Evaluation 5 Quantification

Calibration Curve Fits

The mathematical problem is to find the calibration coefficients in a given curve type which will allow the best determination of a future unknown sample.

The least squares method uses the analytical function data from Use Wavelengths of all standards to determine the calibration coefficients of the chosen calibration curve type by a least squares calculation.

If each ith data set of the n standard data sets is expected to obey a function in the p coefficients, kj, although the real (measured) values may cluster around the function because of statistical errors, i, then the general equation is:

(9)

where

i is 1, 2, 3, n (the total number of standards)

The calibration coefficients, kj, can be estimated using the least squares method, that is minimizing the sum of the squares of the errors, i (the differences between the measured value and the calibration curve).

(10)

In matrix notation:

(11)

where

C is n- concentration column vector

F is n p function result data matrix

k is p- calibration coefficient column vector

is n- error column vector

The elements used in the calibration matrix F and the coefficient vector k are given in Table 8 on page 46 with dimension p of the coefficient vector.

fi ci( , )

ci k0 k1fi k2fi 2 i+ + +=

cactuali ccalculatedi( )2

i 1=

n

i 2

i 1=

n

minimum= =

C Fk +=

Understanding Your UV-visible Spectroscopy System 45

5 Evaluation Quantification

Example

Using curve type III, where p = 2, for 5 standards, n = 5:

through to

That is:

or, in matrix notation:

Table 8 Elements used in calibration matrix F

Curve Type p F(i)th row k

I 1 fi k1

II 2 1 fi k0 k1

III 2 fi fi 2 k1 k2

IV 3 1 fi fi 2 k0 k1 k2

c1 k1f1 k2f1 2 1+ +=

c1 k1f5 k2f5 2 5+ +=

ci c5

f1 f1 2

f5 f5 2

k1

k2

i 5

+=

C Fk +=

46 Understanding Your UV-visible Spectroscopy System

Evaluation 5 Quantification

By defining F and k for each of the calibration curve types, the coefficients are given by the general equation:

(12)

where

FT denotes the transpose of F

Example Continued:

k F T F( )

1 F T C=

k1

k2

f1 f5

f1 2 f5

2

f1 f1 2

f5 f5 2

1

f1 f5

f1 2 f5

2

c1

c5

=

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5 Evaluation Quantification

Calibration Results

This section contains the equations used in the calculations of the results given in the Calibration Table and the Calibration Results Summary.

Residual

(13)

%Error

(14)

where

is the residual of the ith standard (see Residual on page 48)

Std.Dev of Calibration

(15)

where

is the residual of the ith standard

n is the number of standards

p is the number of coefficients in the equation of the calibration curve

ei Cactuali Ccalculatedi=

di ei

Ccalculatedi --------------------------- 100=

ei

s 1 n p( )

---------------- ei 2

i 1=

n

=

ei

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Evaluation 5 Quantification

Std.Dev of Coefficient k

(16)

where

is the jth diagonal element of

s is the standard deviation of calibration (see Std.Dev of Calibration on page 48)

Correl. Coeff (R^2)

(17)

In the case of curve types II and IV,

(18)

In the case of curve types I and III, is set to zero.

skj s F T F( )jj

1 =

F T F( )jj

1 F T F( )

1

R 2

Ccalculatedi C( ) 2

i 1=

n

Cactuali C( ) 2

i 1=

n

-------------------------------------------------------=

C 1 n -- Ci i 1=

n

=

C

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5 Evaluation Quantification

Quantification of Unknown Samples

When the standards have been measured, and the calibration coefficients have been determined, the unknown concentration of a measured sample can be calculated simply by calculating in the equation of the

calibration curve using the

Manualsnet FAQs

If you want to find out how the ChemStation Agilent works, you can view and download the Agilent ChemStation Software User Manual V2 on the Manualsnet website.

Yes, we have the User Manual for Agilent ChemStation as well as other Agilent manuals. All you need to do is to use our search bar and find the user manual that you are looking for.

The User Manual should include all the details that are needed to use a Agilent ChemStation. Full manuals and user guide PDFs can be downloaded from Manualsnet.com.

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You can download Agilent ChemStation Software User Manual V2 free of charge simply by clicking the “download” button in the upper right corner of any manuals page. This feature allows you to download any manual in a couple of seconds and is generally in PDF format. You can also save a manual for later by adding it to your saved documents in the user profile.

To be able to print Agilent ChemStation Software User Manual V2, simply download the document to your computer. Once downloaded, open the PDF file and print the Agilent ChemStation Software User Manual V2 as you would any other document. This can usually be achieved by clicking on “File” and then “Print” from the menu bar.