Steve Bouffard, PhD Agilent Technologies

Product Specialist

Molecular Spectroscopy

Innovations in FTIR and UV Technology for the Rapid Analysis of Liquid Samples

The Agilent Cary 630 FTIR • An entry level FTIR that has changed the way analysts approach

routine infrared measurements

• The World’s smallest FTIR!

• Designed for a wide range of markets, in particular, chemicals QA/QC, pharma QA/QC and teaching

The Agilent Cary 630 FTIR sampling flexibility Solving problems through innovation…

630 Engine DialPath TumblIR

Diamond ATR Diffuse Reflectance Transmission

The traditional approach to liquids analysis- the demountable cell

• Great skill and care is needed to assemble and fill the cell without leakage.

• Cleaning cell in-between each analysis is tedious and laborious.

• 4 samples/hour if you’re lucky

Analagous to ATR sampling with enhanced sensitivity due to increased pathlength

1. Place Sample on

Lower Window

3. Scan the sample

2. Rotate into place

4. Cleaning is easy!

The Agilent Cary 630 approach to liquids analysis- the DialPath and TumblIR sampling modules

The Agilent Cary 630 approach to liquids analysis The TumblIR and the DialPath sampling modules – single pathlength liquid transmission accessory.

A REVOLUTION IN LIQUIDS ANALYSIS

100 μm fixed pathlength

3 pathlengths provide flexibility (30 to 250 μm)

Liquid transmission sampling geometry 3 µ L Fill Volume

250 µ L Fill Volume

µ L Fill Volume

250 µ L Fill Volume

The combination of class-leading performance and rapid scanning capability means that most liquid samples can be measured in < 30 secs. Volatile liquids require > 120 secs. to show any appreciable change in concentration, once placed in the DialPath cell that is in the closed position.

The Agilent Cary 630 approach to liquids analysis DialPath – 3 pathlength liquid transmission cell

• 30, 50, 100 μm fixed pathlengths

• 50, 100, 200 μm fixed pathlengths

• Custom pathlengths available

Pathlength (μm) Typical Conc. Range

30 Neat - 0.1%

50 50% - 500 ppm

100 20% - 100 ppm

200 10 % - 50 ppm

Cary 630 approach to liquids analysis

The DialPath sampling module- 3 choices

ID and Quant of Liquid Raw Materials (FDA)

The challenges for this particular application were:-

1. To confirm the identities of various incoming viscous liquid raw materials (Qualitative)

2. To determine the levels of impurities in these raw materials (across a large concentration range) according to FDA guidelines (Quantitative)

3. To keep the analysis as simple as possible for non-skilled operators, using the minimum number of accessories & measurements for the results

Method

Cary 630 FTIR

Accessory – The DialPath

Collection Conditions: – Resolution: 4 cm-1 – # scans: 64 (~30 sec) – DialPath accessory pathlength:

• 30 um for qualitative • 75 um for quantitative

Results – raw material identification (qualitative) Spectra collected with 30um DialPath setting

glycerol

propylene glycol

dipropylene glyco

triacetin

Spectra are searched against reference database

Results – determination of impurities (quantitative)

Spectra collected with 75um DialPath setting

ethylene glycol in glycerol diethylene glycol in glycerol

R2=0.9895 R2=0.9745

Limit of detection – 0.04% Limit of detection – 0.02%

This fits well with FDA limits of <0.1% impurities in glycerol

Results – determination of impurities (quantitative) The Agilent MicroLab software displays analysis results for the level of ethylene glycol impurity in glycerol. Red color shows that the level of impurity is outside of specification.

Monitoring gasoline in diesel fuel

Figure 1. The IR spectral overlay of gasoline (Blue) and diesel fuel (Red) using the Agilent 5500t FTIR spectrometer, 100 μm pathlength

The chemical differences between gasoline and diesel fuel can be seen in their infrared spectra. Below is a comparison between the infrared spectra of gasoline and diesel fuel highlighting the ethanol and aromatics in gasoline. Neither ethanol nor light aromatics are observed in the diesel fuel spectrum.

Figure 2. The IR absorbance vs. concentration plot for gasoline in diesel fuel, 100 um pathlength

Gasoline absorbance versus concentration plot using gasoline-spiked diesel fuel

UV/Vis Sampling Innovations 1. Fiber optic probe analyses 2. Ultra-microvolume cuvettes Increase productivity… Lower cost of analysis

The Cary 60 UV/Vis system is optimized by design for fiber optic probe analyses • High energy Xenon flash lamp coupled with low-noise

electronics provides uncompromised data quality

• Room light immunity minimizes stray light effects

• Small focused beam provides superior coupling to fiber

What are Fiber Optics?

• A fiber optic probe is essentially a light guide that takes the UV-Visible light from the instrument into a fiber – to the sample – and then back to the instrument.

• Performance depends on: • Efficiency of coupling the light from the instrument to the fiber • Ability to shield the fiber tip from the stray light effects for ambient (room)

light.

• Most commercial spectrophotometers have poor coupling (due to large beam size) and are not room-light immune, thereby, making the use of fibers impractical.

Why use Fiber Optics? No more need for expensive cuvettes! Improve workflow and increase productivity by taking the instrument to the

sample Measure cold, hot, toxic/radioactive, odd-shaped samples Measure samples in situ

Measure cold samples directly from the

refrigerator!

Improve workflow and minimize

sample preparation

Fiber optic couplers for Cary 60 UV/Vis system

The fiber optic dip probe is a trouble-free alternative to a conventional sipper.

The fiber optic coupler allows you to connect a fiber optic probe to suit your application.

Measuring the nitrate content in water

Challenges: Solution: Benefits:

American Public Health Association (APHA) Nitrate method Rapid measurement – high sample throughput Simple measurement and auto report generation

Cary 60 with fiber optic dip probe

Room light immunity enabling simple sampling with fiber optics WinUV software for method creation Control instrument from sampling accessory

Concentration and Fiber Optics

Using fiber optic dip probe reduced

time of analysis by 50% Standards curve shows excellent

linearity for accurate measurements of unknowns.

Excellent repeatability on Raw Abs

data demonstrates the performance of the F.O. probe.

Measuring nitrate levels in waste water

Application Note Publication Number 5990-7932EN

Sample Conc mg/L

Mean Abs

SD %RSD Raw Abs

A 0.145 0.0510 0.0009 1.78 0.0520 0.0504 0.0506

B 0.709 0.1825 0.0025 1.36 0.1797 0.1838 0.1841

Measuring the nitrate content in water

Investigating the photochemical properties of Methylene Blue

Challenges: Solution: Benefits:

To analyze the changes, in situ, of Methylene Blue (a photosenstive compound used in cosmetics and sunscreen products) upon its exposure to UV radiation. UV/Vis systems with continuous sources degrade the sample

Cary 60 with fiber optics probe

Unique Cary 60 flashlamp does not cause photodegradation Ability to measure the sample out of the instrument Fastest scan rate to obtain multiple spectra over a short time period

Investigating the photochemical properties of methylene blue

Scanning kinetics

Measuring photo degradation of methylene blue exposed to high intensity UV light from external

source

Application Note Publication Number 5990-7864EN

Challenges: Solution: Benefits:

To measure biological sample directly from storage (eg. fridge) – without compromise to data quality - saving time and increasing sample throughput.

Cary 60 with fiber optics microprobe

Save time and money – increase no. of samples measured and use significantly less sample volume (< 40 µL) Improve workflow by minimizing sample handling and transfer to cuvettes No compromise in accuracy or reproducibility of data due to unique optical design

Measuring low volumes of DNA at 4°C

Scans of 150 µl samples of DNA at 4 ºC at 2 concentrations showing the characteristic absorbance peak at 260 nm. Note peak absorbance of 1.0 absorbance units for 50 µg/ml DNA versus peak absorbance of 0.5 absorbance units for 25 µg/ml DNA demonstrating linear adherence to the Beer Lambert Law (A= εcl)

Application Note Publication Number 5990-7863EN

Measuring low volumes of DNA at 4°C

The ultra-microvolume cuvette Analogous to ATR use in the FTIR Fiber optic cables in a cuvette Microliters or less of sample needed

Factor 2 mm Cap (ng/µL)

1 mm Cap (ng/µL)

0.2 mm Cap (ng/µL)

0.1 mm Cap (ng/µL)

Total Detection Range (ng/µL)

Factor 5 10 50 100

dsDNA 50 6-425 *2-850 65-4250 125-8500 6-8500

ssDNA 37 5-315 10-630 50-3145 95-6290 5-6290

RNA 40 5-340 10-680 50-3400 100-6800 5-6800

Oligo 33 4-280 8-560 45-2800 85-5610 4-5610

Sample Volume

6 - 10 µL 3 - 5 µL

0.7 - 4 µL

0.5 - 3 µL

Cary 60 with Ultra Microvolume Cuvette

*tested on a Cary 60 Manufacturer recommendations

Challenges: Solution: Benefits:

Measure 4 µL of sample accurately and reproducibly

Cary 60 with ultra-microvolume cuvette Direct measurement at 280nm Plot Abs at 280 vs. concentration using standards Accurate and reproducible results Minimize dilutions and reduce sample preparation errors Preservation of precious/expensive samples

Protein/Nucleic Acid Conc. – Low Sample Volume

Protein Concentration – Low Sample Volume

Multiple wavelength scans of DNA demonstrate the superior reproducibility of the Cary 60

using only 4 µL of DNA sample!

Summary • The Agilent Cary 630 FTIR, in addition to being the World’s

smallest FTIR has unique liquid transmission sampling modules- the DialPath and TumblIR. Transmission sampling provides the ease of ATR with increased sensitivity

• The Agilent Cary 60 UV/Vis System is optimized for fiber optic analyses and for ultra low volume liquids analyses. Its small beam geometry is the key to providing the best sensitivity for these techniques