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8453
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16
Theory of Operation
This
chapter
has four s
ections:
•
“
Instrument Ov
erview
”
on page
18,
•
“
The
ory o
f Operat
ion an
d Cont
rol
”
on page 28,
•
“
External
Commun
ication
”
on page
50, an
d
•
“
Inte
rnal Connec
tions
”
on pag
e 62.
121
123
Table of Contents
Main Page
4 Using Your Agilent 8453 Uv-Visible Spectroscopy System
55
Table of Contents
55
Starting Your First Measurement Session
56
Starting Your UV-Visible Software
58
Measuring Caffeine Absorbance at 273 Nm
59
Saving Your Parameters as a Method
62
Retrieving and Printing a Method
64
Saving and Retrieving Data
67
Print Preview of Reports
73
Finding the Caffeine Absorbance Maximum
76
Entering Your Cell's Path Length
80
Controlling Your Sipper System
81
Using Your Multicell Transport
83
Quantitative Analysis Using a Calibration with Standards
86
How Can I be Sure that My Agilent 8453 Works Properly
93
How Can I Get a Deeper Understanding of UV-Visible Spectroscopy
96
When Do I Have to Measure a Blank
98
Table of Contents
112
Default Chapter
162
External Connectors
162
Internal Connections
168
Connector Definitions
169
3 Diagnostics and Troubleshooting
173
Explainations of Status and Error Messages and a Logical Approach to Troubleshooting
173
Front Panel Status and Power Switch Leds
175
Error Messages
177
General Troubleshooting Hints
186
4 Maintenance and Repair
199
Procedures for Exchanging Parts, such as Lamps and Electronic or Mechanical Items, and for Cleaning Lenses
199
Maintenance
200
Cleaning the Instrument
201
Exchanging the Deuterium or Tungsten Lamp
202
Cleaning the Stray Light Filter
207
Cleaning the Lenses
209
Repair Procedures
214
Removing and Replacing Covers
215
Exchanging Keyboard and Key Pad
222
Exchanging or Upgrading Internal Memory
224
Exchanging the SPM Board
226
Exchanging the Optical Unit
228
Exchanging the Shutter Assembly
230
Exchanging the Fan Assembly
232
Exchanging SDA Board
234
Exchanging SLS Board
236
Exchanging the Main Power Supply
239
5 Parts and Materials
243
Exploded Views of Repairable Parts and Part Number Listings for Ordering Replacement and Exchange Parts
243
Exploded Views and Part List
244
6 Interfacing
251
Communicating and Interfacing through GPIB and RS-232C
251
Setting the 8-Bit Configuration Switch
252
Table of Contents
279
1 Agilent Chemstation Features
284
General Description
285
Chemstation Hardware
288
About the Chemstation Software
289
Instrument Control
305
Documentation
306
The Chemstation Directory Structure
308
Navigation Pane
312
2 Methods
315
What Is a Method
316
Parts of a Method
317
Method Information
317
Instrument Control
317
Data Analysis
318
Run-Time Checklist
319
Status of Methods
320
Stored Methods
320
Current Method
321
Creating Methods
322
Editing Methods
323
Method Parts to Edit
323
Method Directory Structure
325
What Happens When a Method Is Run
326
Method Operation
326
Pre-Run Command or Macro (Run Time Checklist)
327
Data Acquisition (Run Time Checklist)
327
Data Analysis (Run Time Checklist)
328
Customized Data Analysis (Run Time Checklist)
329
Save GLP Data (Run Time Checklist)
329
Postrun Command or Macro (Run Time Checklist)
330
Save Copy of Method with Data (Run Time Checklist)
330
Save Copy of Method as DA.M with Data (Chemstation Default)
330
Method Operation Summary
331
3 Data Acquisition
333
3 Data Acquisition
334
What Is Data Acquisition
334
Data Files
335
Online Monitors
337
Online Signal Monitor
337
Online Spectra Monitor
337
Logbook
338
Status Information
339
Chemstation Status
339
Status Bar
339
System Diagram
340
4 Integration
341
What Is Integration
343
What Does Integration Do
344
The Chemstation Integrator Algorithms
345
Integrator Capabilities
345
Overview
347
Defining the Initial Baseline
347
Tracking the Baseline
348
Allocating the Baseline
349
Identifying the Cardinal Points of a Peak
350
Definition of Terms
351
Cardinal Points
351
Solvent Peak
351
Shoulder (Front, Rear)
352
Slope
352
Principle of Operation
353
Peak Recognition
354
Peak Width
354
Peak Recognition Filters
355
Bunching
356
The Peak Recognition Algorithm
357
Non-Gaussian Calculations
360
Baseline Allocation
363
Default Baseline Construction
363
The Start of the Baseline
364
Tick Marks
364
The End of the Baseline
364
Baseline Penetration
365
Peak Valley Ratio
366
Tangent Skimming
368
Unassigned Peaks
373
Peak Separation Codes
373
Peak Area Measurement
376
Determination of the Area
376
Units and Conversion Factors
378
Integration Events
379
Initial Events
379
Timed Events
382
Autointegrate
382
Manual Integration
384
5 Quantification
387
5 Quantification
388
What Is Quantification
388
Quantification Calculations
389
Correction Factors
390
Absolute Response Factor
390
Multiplier
390
Dilution Factor
390
Sample Amount
391
Uncalibrated Calculation Procedures
392
Area% and Height
392
Calibrated Calculation Procedures
393
ESTD Calculation
394
Norm% Calculation
396
ISTD Calculation
397
Run 1: Calibration
398
Run 2: Unknown Sample
398
ISTD Calculation of Calibrated Peaks
399
ISTD Calculation of Uncalibrated Peaks
399
6 Peak Identification
401
6 Peak Identification
402
What Is Peak Identification
402
Peak Matching Rules
403
Types of Peak Identification
404
Absolute Retention/Migration Time
404
Corrected Retention/Migration Time
404
Peak Qualifiers
404
Amount Limits
405
Absolute Retention/Migration Time
406
Corrected Retention/Migration Times
408
Single Reference Peaks
408
Multiple Reference Peaks
408
Peak Qualifiers
410
Signal Correlation
411
Qualifier Verification
411
Qualifier Ratio Calculation
411
The Identification Process
413
Finding the Reference Peaks
413
Finding the ISTD Peaks
413
Finding the Remaining Calibrated Peaks
414
Classification of Unidentified Peaks
414
7 Calibration
415
7 Calibration
416
Definition of Terms
416
Calibration Table
417
Calibration Curve
418
Unknown Samples
420
Types of Calibration
421
Single-Level Calibration
421
Multilevel Calibration
422
Calibration Ranges
424
Calibration Curve Fits
424
Origin Treatment
424
Group Calibration
427
Peak Summing
428
Recalibration
429
What Is Recalibration
429
Why Recalibrate
429
Manual Recalibration
429
Recalibrations with Peak Summing
430
Recalibration Options
430
Ways to Recalibrate
431
Recalibration of Unidentified Peaks
431
8 Automation
433
8 Automation
435
What Is Automation
435
What Is a Sequence/Sequence Template
436
Preferences - Sequence Tab
437
Sequence Parameters
439
Sequence Table
440
Creating Sequences (Sequences and Sequence Templates)
441
Using the Sequence Table Editor
441
Using the Insert Vial Range Button
441
Using the Append Line Button
441
Using the Custom Fields Button
442
Working with Sequences (Sequences and Sequence Templates)
443
Priority Samples
443
Sequencing with Control Samples
443
Stopping a Sequence
443
Aborting a Sequence
444
Pausing a Sequence
444
Running a Partial Sequence
444
Sequence Log File
446
What Happens When a Sequence Is Run
447
Sequence Data File Structure (Unique Folder Creation ON)
449
Data File Naming in a Sequence
450
Automatic Data File Naming During a Sequence
450
Entering Data File Names Manually
451
Postsequence Operation
452
Not Ready Timeout (LC and CE Only)
452
Wait Time (LC and CE Only)
452
Automatic Recalibration
453
Specifying Recalibrations
454
Recalibration Parameters in the Sequence Table
454
Types of Sequences
457
Explicit Calibration Sequences
458
Cyclic Single-Level Calibration Sequences
459
Cyclic Multiple-Level Calibration Sequences
460
Method a Analysis Order
461
Method B Analysis Order
462
Explicit and Cyclic Calibrations Together
464
Example
464
Simpreg Analysis Order
465
Cyclic Calibration Sequences with Bracketing
466
Example
466
Bracketed Sequence Operation
467
Example
467
Cyclic Recalibration Sequences with Multiple Vials Containing the same Dilution of a Standard
470
Cyclic Recalibration Sequence with "Round-Robin" Calibration Vial Usage
470
Cyclic Recalibrations Where each Calibration Uses a Different Vial
472
Bracketing Sequence that Uses Different Vials for Opening and Closing Bracket
472
9 Data Review, Reprocessing and Batch Review
475
9 Data Review, Reprocessing and Batch Review
476
Navigation Table in Data Analysis
476
Navigation Table Configuration
476
Navigation Table Toolbar
477
Data Review Using the Navigation Table
478
Sequence Reprocessing Using the Navigation Table
479
What Is Batch Review
481
Enabling Batch Review Functionality with Chemstation Openlab Option Installed
482
Batch Configuration
483
Batch Table
483
Compound Table
483
Batch Report
484
User Interface
484
Review Functions
485
Calibration in Batch Review
485
Batch Reporting
486
Batch History
486
10 Using the Chemstation Reports
487
10 Using the Chemstation Reports
488
What Is a Report
488
Reporting Results
489
Uncalibrated Reports
489
Calibrated Reports
489
External Standard Report
489
Internal Standard Report
490
Control Charts Report
490
Quantitative Results
491
Reporting Custom Field Values
493
Report Styles
494
Adding a Customized Report to Report Styles
496
Other Report Style Parameters
497
Summed Peaks Table
497
Report Layout for Uncalibrated Peaks
497
Report Destination
498
Report File Formats
498
Sequence Summary Reporting
500
Overview
500
Setting up a Sequence Summary Report
500
11 Evaluating System Suitability
505
11 Evaluating System Suitability
506
Noise Determination
509
Noise Calculation Using Six Times the Standard Deviation
509
Noise Calculation Using the Peak-To-Peak Formula
510
Noise Calculation by the ASTM Method
511
Signal-To-Noise Calculation
513
Drift and Wander
513
Calculation of Peak Symmetry
514
System Suitability Formulae and Calculations
516
General Definitions
517
Void Volume
517
Retention Time of Unretained Compound T (M) [Min]
517
Performance Test Definitions
518
Statistical Moments
518
Statistical Moments, Skew and Excess
519
True Peak Width Wx [Min]
520
Capacity Factor (USP), Capacity Ratio (ASTM) K
520
USP Tailing Factor (USP) T
520
Number of Theoretical Plates Per Column (USP, ASTM) N
521
Number of Theoretical Plates Per Meter N [1/M]
522
Relative Retention (USP, ASTM), Selectivity Alpha
522
Resolution (USP, ASTM) R
523
Definitions for Reproducibility
524
Sample Mean M
524
Sample Standard Deviation S
524
Relative Standard Deviation RSD[%] (USP)
525
Standard Deviation of the Mean SM
525
Confidence Interval CI
526
Regression Analysis
527
Internally Stored Double Precision Number Access
529
12 System Verification
533
Verification and Diagnosis Views
534
System Verification
534
The Glpsave Register
537
DAD Test Function
539
Review DAD Test Function
539
Table of Contents
550
1 Chemstation Data Structure
551
Chemstation Prior to B.02.01
552
Chemstation B.02.01 and Higher
553
2 Data Acquisition
564
3 Data Analysis
568
Data Analysis: Data Review
569
The Chemstation User Interface During Data Review
576
Data Analysis: Reprocessing Data
578
4 Workflow with Unique Folder Creation Switched off
582
Working with "Unique Folder Creation" on or off
582
Workflow with Unique Folder Creation Switched off
583
Sequence Container Migration
587
Table of Contents
595
1 Installation and Configuration
597
Installing the Biochemical Analysis Software
598
Configuring Your Agilent 89090A Peltier Temperature Controller
599
2 Kinetics Mode
601
Preparing a Time Based Measurement
602
Starting the Kinetics Mode
602
Setting up Your Spectrophotometer
604
Configuring the Sampling System
606
Setting up a Kinetic Method
608
The Kinetic Measurement
613
Running a Time Based Measurement
613
Evaluating Your Kinetic Data
614
Viewing Results
616
3 Thermal Denaturation Mode
617
Preparing a Thermal Denaturation Measurement
618
Starting the Thermal Denaturation Mode
619
Setting up Your Spectrophotometer
620
Setting up the Temperature Ramp
620
Setting up a Thermal Denaturation Method
621
The Thermal Denaturation Experiment
626
Running a Thermal Denaturation Experiment
626
Evaluating Your Thermal Denaturation Data
627
Viewing Results
628
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