selecting the correct spectrophotometer for quality results · •optical order sorting filters are...

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 Jim.Steensrud@Agilent.com

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My Contact is Jim.Steensrud@Agilent.com

Thanks Again