selecting the correct spectrophotometer for quality results · •optical order sorting filters are...
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Selecting The Correct Spectrophotometer ForQuality Results
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CAGPresented by Jim SteensrudApplications EngineerAug 2012
ChemicalsChemicals - Applications Overview• QA/QC on raw materials and finished product in manufacturing
• Chemical identification or study of chemical processes•Synthetic chemistry labs, Photochemistry research, Nanoparticle characterization, Surface chemistry research, Analytical chemistry
• Color measurements• Color measurements• Solutions and films (color matching, UV protection of sunglasses)
Wavelength Scans – What is it? Concentration – How much? Kinetics – what is occurring?
Optical Spectroscopy Measurement Attributes• Versatile – solids, liquids and gases
• Sensitive - from micro gram to tens of percentages of analyte
• In addition to concentration many other properties can be measured
• Chemical rates
• Thermodynamic properties
• Bond angles• Bond angles
• Polarization states
• Transmittance and reflectance qualities
• Color transforms and matching
• Electronic band gaps
• Sample impurities
• Several more relationships
One Of The Most Versatile Analytical Techniques
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Electromagnetic Spectrum
Basic Electromagnetic Theory
Electric
Magnetic
Electromagnetic energy propagates through space as two oscillating fields oriented perpendicular to the direction of propagation and to each other.
These fields can interact with matter.
Electronic Vector Used
Electric Vector
Wavelength (nm) or Frequency (cm-1 )
Frequency related to wavelength by νννν = c/λλλλ.
• λ = Wavelength, in cm.
• c = Speed of Light.
Energy related to frequency by E = hνννν.
• ν = frequency, Hz.
• h = Planck’s Constant.
How Does Optical Spectroscopy Work From The Chemistry (1)
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How Does Optical Spectroscopy Work From The Chemistry(2)
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The Definitive Measurement For The Effect Of SBW on Peak Height
Observed Peak Height
Observed Peak Height
True Peak Height
True Peak Height
1.01.0
0.90.9
0.80.8
0.70.7
0.60.6
Observed Peak Height
Observed Peak Height
True Peak Height
True Peak Height
Spectral Band WidthSpectral Band WidthNatural Band WidthNatural Band Width
0.1 0.2 .4 0.6 1.0 2 4 6 80.1 0.2 .4 0.6 1.0 2 4 6 8
0.60.6
0.50.5
0.40.4
0.30.3
Rule Of Spectral Band Width (SBW)
SBW = One Tenth of the Natural Bandwidth
Results in a Deviation of <0.5 % of the Results in a Deviation of <0.5 % of the True Peak Height
Peak Distortion Do Lack Of Resolution
How Cary 100/300 and 4000-6000i Calibrate Wavelength
What’s This Stuff About Resolution?
1.5 nm SBW
0.2 nm SBW
Holmium Oxide Reference Filter
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The Hidden Truth About Resolution
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Wavelength and Abs Error
Hi Res Scan Cary 6000i Two Scans
So, Samples Drive The Instrument Selection
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Criteria For Measurement Success
Performance
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SoftwareCalculations
Sample Handling
Cary Systems
The Sample and Measurement Expectations Dictate The Instrumental Choice
• What are the samples
• Is there a method
• What Spectral Resolution is required
• What wavelength range – UV, Visible, NIR or Mid IR
• What sample handling accessory(s)
• What data reductions and results calculations are required and expected.
• Measure using Absorbance or Fluorescence Spectrophotometers
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Instrument Systems
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UV-Vis NIR Spectroscopy SolutionsThe Agilent Cary Family
Cary 300
Cary 4000
Cary 5000/6000i
Cary Eclipse
Per
form
ance
“A word from our sponsors”
Cary 100
Cary 300
8453
Cary 60
Per
form
ance
Routine to Research
How Does Optical Spectroscopy Work From The Instrument View
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SourceEnergySelection Sample Detector Display
Cary Eclipse Fluorescence Spectrometer
The Power of Xenon…
• Unique Xe flashlamp technology
• Measure small volume samples
• Fiber optics • Fiber optics
• Room light immunity: unique, Varian patented technology
• Eliminates photo-degradation
• Long lamp lifetime
Application focus
• Biochemical applications
• Academia
• Industrial chemistry
Cary Eclipse Fluorescence Spectrometer
• Light source: xenon flash lamp (80 Hz)
• Room light immunity
• Instrument WL range: 200-850 nm (detector limited)
• Ex and Em SBWs (nm): 1.5, 2.5, 5, 10, 20 nm
• Optical order sorting filters are standard• Optical order sorting filters are standard
• Sample and reference detector: R928 PMT
• Max. scan rate 24000 nm/min
• Same “Locked” Scan as all Cary UV-Vis NIR systems
• Extensive array of accessories
Eclipse SBW Control
Cary Eclipse Horizontal SBW Advantage
Feature Advantage Benefit
Horizontal slit orientation
•Increased viewing region
•Increase luminescence signal by factor of > 5 times over vertical slits
•High sensitivity• Less sample volume in
•High quality data
• Less sample volume in standard cell
Cary Eclipse - Room Light Immunity
Raman band
Dilute fluorescein solution
Detector
source S
S:R
sample
Technology Overview Double Beam
Mono chopper
Cary 100, 300, 4000, 5000, 6000i
Detector
R
S:R
reference
access bbbb
Mono chopper
Detector
SourceS
S:R
sample
Technology Overview Dual Beam – Cary 60
Mono
R
reference
Detectorno accessto reference
beam
How Many Beams In The Sample Compartment??The Agilent 8453A and Cary 50 only have one beam in the sample compartment – Can Not do spectral difference in real time. 4 samples of Cytochrome P-450
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Control Data Interval
30
Impurity Data A Typical Assay
Impurity
A Complete Solution For Time Based Measurements!• Many enzymatic reactions can be analyzed by detecting changes in the full spectrum
over time
• These scans performed with 8453 and a single cell Peltier for temperature control –solutions for both time and temperature dependent measurements
(substrate)(substrate)
(product)Isosbestic point
Performance
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High Concentration Samples
Beer’s Law A=a x b x c
2 Abs Is A Myth
Cell Inserts – Attenuate The Sample Pathlength
Whole Blood With Insert
Sample Precision Is Never Better Than The Noise Allows
Solid Sample Different Position
Thickness Measurements Of Solid Sample
Cuvette Selection For Near Infrared Region (NIR)
Cary 60 Photo Sensitivity Zn PorphyrinNo Source Light Loading
80 Flash Per Sec Sample Compartment Was Open For 2 SecIn Lab With Normal Room Light
Dead Volume Mixing Test – “Rapid Mix”
Direct Measurement of Turbid Samples
High Performance UV-Vis NIR Scattering Samples
Scattering Sample With RBA
Low UV Data Standard Nitrogen Purge
Software and Calculations
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Cary Win UV Software Apps
Cary WinUV Instrument Setup
Cary 6000i
Cary 60
Abs vs %T To View Data
Scan Application Has Spectral Calculator
Thin film ADL Program
Scanning Kinetics With Data Extraction
Termal Denaturation Calc (1)
Thermal Denaturation Calc (2)
Color Application Setup
Color Application
Color Calc. Results
Sample Handling
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UV-Vis-NIR Sampling Theory
Sample
Incident Light, Io
UV-Vis-NIR Sampling Theory
UV-Vis-NIR Sampling Theory
UV-Vis-NIR Sampling Theory
UV-Vis-NIR Sampling Theory
Scattering
New Releases - Harrick Accessories
Variable Angle Transmission Accessory
• This accessory is designed to measure samples up to 10 mm thick.
• To overcome beam shift problems which may occur due to measurement angle, sample thickness and refractive index, the VATA allows a sample and blank to be placed in the beam at the same angle of incidence but in the opposite orientation.
• The VATA incorporates a holder for an optional polarizer – both Glan-Taylor and Glan-Thompson are accommodated
• Rear beam attenuation can also be fitted to this accessory.
•One lockdown plate suitable for all instruments
Cary 4000-6000i Absolute Specular Reflectance Accy
Specular Reflectance Measurements
Harrick Praying Mantis – Horizontal Sample Mount
Instrument Beam(s) Are Polarized
Electric Vector
Polarization Study Sample
Inst. Beam S Pol
Sample S Pol
Sample P Pol
Sample Handling Simplified For Vis Measurements
For Cary 60
Masked Cuvettes For Scattering Samples
Cary 60 Differentiators
Fiber OpticsNo more need for expensive cuvettes!Improve workflow by taking the instrument to the sampleMeasure cold, hot and odd-shaped samples
Measure cold
samples
Improve workflow and samples
directly from the
refrigerator!
and minimize sample
preparation
APPLICATION NOTES AVAILABLE:
5990-7863EN Measuring purity of low volumes of DNA at 4 ºC with fiber optics microprobe5990-7945EN Determination of drug tablet concentration in pharmaceutical applications for drug development5990-7864EN Simple, automated measurements of the photocatalytic properties of colorimetric species
Application Focus
DNA scans using fiber optic microprobe
No compromise in data quality, strong linear correlation:
relationship between absorbance and concentration
Abs
DNA concentration (ng/µl)
y = 0.0198xR² = 0.9996
0.0
0.5
1.0
1.5
2.0
0 20 40 60 80 100www.agilent.com (Publication Number 5990-7863EN)
• Easy to clean and load• No sample carry over• Recovery of precious samples• Different path length options• Can be thermostated• Ease of use:
Tray Cell Performance Benefits
Measure High Abs With Short Paths
• Ease of use:• Pipette sample• Place lid over sample• Take measurement• Wipe clean
Tray Cell Accuracy Cary 4000 vs Cary 60
Hellma TrayCell – linearity
Excellent linear performance to 2.3abs
Detect down to 0.001 mg/mL of BSA (using 10mm pathlength cuvette)
Dedicated software
77
Dedicated software
Hellma TrayCell10mm cuvette 1.0mm cap 0.2mm cap
max abs 3.3 abs 2.3 abs 2.3 absdilution factor 10x 50x
equiv abs 23 abs 115 abs
No dilution necessary 0 to 115 abs!
More Options for Measurements of Microvolumesby UV-Vis For customers who need to measure the concentration or purity of small volumes of precious samples such as nucleic acids or proteins - there is a better alternative! With a design focused on ease of use, and superior performance in sensitivity and reliability, the Agilent solution is the Cary 60 and the ultra-microvolume cuvette accessory, which now has more options available for a wider range of sample volumes and concentrations.
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Fun Stuff
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Fun Stuff
Oxford DN Cryostat in Eclipse
Mounts Are Available For Cary 60 – 6000i
Cary Eclipse Solid Sample Holder
Measuring A Single Grain Of Rice
100
150
200
Inte
nsity
(a.
u.)
400 420 440 460 480 500
50
Wavelength (nm)
Four Different Types Of Rice
Cary 50 With Remote DRA and Camera
See The State Names On The Back Of $ 5.00 Bill
Image from on board camera
Size Of The Illuminating Fiber Sizes The Image
Fiber Optic Eclipse
Cary 50/ 60 With Microscope
Cary Instrument Certification
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Instrument Certification
Cary Win UV Testing
Cary 6000i Cary 60
Cary Win UV Photometric Accuracy
Thanks For Your Patience And Good Humor
Shortly You Will Receive A LinkTo Down Load The Presentation
My Contact is [email protected]
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My Contact is [email protected]
Thanks Again