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Nous desenvolupments i aplicacions

UV-Vis /FTIR

Marisa Lleberia.

marisa_lleberia@agilent.com

Account Manager

Espectre UV-Vis

10 200 400 500 100 300 600 700 800

Vio

let

Blu

e

Gre

en

Yel

low

Ora

ng

e

Red

Fa

r U

V

Nea

r U

V

Far UV 10 - 200 nm

Near UV 200 - 380 nm

Visible 380 - 780 nm

Near IR 780 - 3200 nm

Wavelength (nm)

Espectofotémtres tenen el seu Rang Espectral , molt habitual 190 a 1100nm

Totes les mol.lècules orgànicas son capces d’absorbir radiació en aquesta zona de l’espectre.

Modes de Mesura

% Transmisió

Absorbància A.U

Relació I/I0 =

Transmitància

Absorbànciaλ = - ln (I/I0)

I0 I

A = ε b c

Llei Lambert Beer

A = Absorbància

ε = Coeficient absorció

b = Camí óptic cel.la

c = Concentració

Família Agilent UV-Vis/NIR

Price

P

e

r

f

o

r

m

a

n

c

e

$30k $50k

Cary 300 Cary 4000

8453

Cary 100

$8k $80 - 100k

Cary 5000/6000i

Cary 60

Espectofotómetres UV-Vis

5

• Feix únic

• Doble feix Tipo 1

• Doble feix Tipo 2

• Doble Feix Real

--------------------------------------

• Diode Array

Monochromator

Sample

Detector (Si-Diode)

Espectofótometre feix únic

Source

lamps

Performance is compromised by

any drift in source intensity. Not

stable over long periods of time.

No disponibles Agilent !

Reference

Sample

Doble Feix Tipo 1

Monochromator

No disponible a Agilent !

Beam-Splitter

(Ratio: 50/50)

Source

lamps

Detector (Si-Diode)

Detector (Si-Diode)

Doble Feix Tipo 2 ( moderns)

Sample

Beam-Splitter

(Ratio: 50/50)

Sample

Detector

Agilent Cary 60

Source

lamp

Reference

Detector

Detector (PMT)

Reference

Sample

Chopper as a

Beamsplitter

Espectofotómetre doble feix real

Monochromator

Agilent Cary 100/300 and 4000 to 6000i

Source

lamps

100% Light

100% Light

Espectofotómetre Diode Array

Sample

Diode-Array-Detector (or

CCD detector)

Agilent 8453

Source

lamps

Espectrofótometre 8453 UV-Vis PDA

• Característiques:

• 190 -1100nm Wavelength Range

• 1nm Slit Width, 1nm Sampling Interval

• Full Spectrum within 0.1 Second

• Perfect for Ultra Fast Experiments

• Controlled by the Agilent ChemStation Software

• Dissolution Software Driver Available

• Fully EP and USP Compliant

8450

Introduced 1979

Agilent (HP) UV-Visible Spectroscopy

Systems Spectrophotometer History

8451

Introduced 1984

8452A

Introduced 1986

8453

Introduced 1995

• Xenon Flash Lamp Source

• Wide Wavelength Range 190 – 1100nm

• Fixed 1.5nm Bandwidth.

• Room Light Immunity

• Focussed Beam

• Wide Range of Accessories

Espectrofotómetre Cary 60

Cary 60 – Lampara Xenón

• Xenon Flash Lamp Source

3 x 109 flashes typical lifetime – 80 flashes/secon

ONLY flashes during measurements so lamp lifetime can be

considered to be well in excess of 10 years for most working labs,

and we offer 3 years warranty on the lamp as standard!

Característiques Generals – Flash Xenón

Broad excitation range with

high efficiency so intensely

bright - typically more than one

order of magnitude brighter than

D2 and Halogen lamps used in

conventional double beam

instruments.

The lamp needs NO

WARMUP TIME

Focussed Beam

Xenon Flash Lamp Source – Fast Data Collection

80 flashes/second means that we can collect 80 data

points/seconds, and at maximum scan speed of

24,000nm/min. This allows us measure the kinetics of very

fast reactions.

Complete spectral scan takes <3 secs.

1.8 seconds

Accessoris Cary 60

Application:

Challenges:

Solution:

Benefits:

To measure biological sample directly from storage

(eg. fridge) – without compromise to data quality

Measuring cold samples (4 C)

Cary 60 with Fiber Optics Microprobe

Save time and money

Improve workflow by minimizing sample

handling and transfer

No compromise in accuracy or

reproducibility of data due to unique optical

design

Exemple 1: Cary 60 amb Fibra

òptica

Exemple 2: Sistema automàtico On-line UV-Vis

Configuración con sistema de medida por Fibra

Flexibilitat tria caminos ópticos de 1 mm a 20 mm

Medidas con intervalos de 30 seg.

Elimina cualquier tipo de contaminación

Page 19

Exemple 3 : Micro Tray Cell Puressa de Proteina/

Acid Nucleic (DNA/RNA)

APPLICATION NOTES AVAILABLE

SI-A-1219 Practical Limits of DNA Quantitation in Microliter samples

5990-7863EN Measuring the purity of low volumes of DNA at 4 ºC with fiber optics microprobe

It is common for nucleic acid solutions

to be contaminated with other

molecules such as proteins, phenol

etc

Proteins absorb at 280nm

DNA/RNA absorbs at 260nm

To assess purity we use the ratio of

260:280nm

Pure DNA has a 260:280 ratio of ~1.8

Pure RNA has a 260:280 ratio of ~2.0

Pure proteins will give a value

between 0.5 and 0.7

Exemple 4: Suport de cel.la termostatitzat.

Cinétiques enzimàtiques

Enzymes are naturally produced

proteins that act as catalysts to drive

biological reactions, so the study of

enzyme reactions is a key part in

understanding the biochemistry of

life.

In this type of study, the enzyme

solution is usually held in a cuvette

and the substrate solution is pipetted

into the cuvette. The temperature of

the solution is important, so these

reactions are normally monitored

using a thermostatted cell holder, and

the solutions are stirred using a

magnetic stirrer.

Using either fixed wavelengths,

or a series of spectral scans, the

reaction is monitored over time.

Exemple 5 : Espectofotómetre On-line

Configuración con

Transportador Multi-Celda

y bomba peristáltica.

El Transportador

Multicelda de 16 posiciones

del Cary-60 permite

conectar 2 Baños de forma

simultanea

Page 22

Exemple 6 : Reflactància Difussa. Mesura mostres

sólides. Application:

Challenges:

Solution:

Benefits:

Rapid Grading of Pearl Quality

To quickly and simply measure the “lustre” and

“colour” of jewellery grade pearls

Cary 60 with Barrelino remote diffuse reflectance

accessory

Save time and money

Reduce “subjective” nature of the

tests

Can be done by “unskilled” labour

Still the leader

in Xenon flash lamp

technology after 12 years!

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Altres exemples d’aplicació:

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IR

IR: Provoca movimientos vibracionales y rotacionales en las moléculas con cierto momento dipolar.

• Permite identificación precisa de los enlaces

• Excelente herramienta para identificar estructuras moleculares

• Rangos:

– NIR; 4.000 cm-1 a 14000 cm-1 (2.500 nm a 700 nm)

– Mid-IR; 4.000 cm-1 a 400 cm-1 (2.5 µm a 25 µm )

– FIR; 400 cm-1 a 10 cm-1 (25 µm a 1000 µm )

ΰ = 1

E = h c ΰ

(cm-1)

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Interferòmetre

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Zones de l’espectre infrarroig

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Frequències enllaços

Per què podem utilitzar FTIR?

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Ethylene glycol

4000 3000 2000 1000

3.0

2.0

1.0

0.0

Wavenumber

Ab

so

rban

ce

Spectrum from an Unknown Sample

4000 3000 2000 1000

3.0

2.0

1.0

0.0

Wavenumber

Ab

so

rban

ce

Comparació amb

espectres d’una base

de dades

Gluestick

4000 3000 2000 1000

1.0

0.6

0.2

Wavenumber

Ab

so

rban

ce

Polystyrene

4000 3000 2000 1000

8

6

4

2

0

Wavenumber

Ab

so

rban

ce

Anàlisi Qualitatiu

Espectroscopia Infraroja es una

técnica ideal per a identificació

compostos desconeguts

• FT-IR pot ser utilitzat per anàlisi quantitatiu seguint llei de

Beer-Lambert law.

• La corba de calibració es un gràfic de la resposta

(absorbància) vs concentració d’un etsandard..

• Permet mesures simultàneas

-0.012

-0.006

0.000

0.006

0.012 A

.U.

1000 2000 3000 4000

Wavenumbers (cm-1)

Per què podem utilitzar FTIR?

Anàlisi Quantitatiu

FTIR 630

• An entry-level FTIR that will change the way our customers approach

routine infra-red measurements!

• Complements our 600 Series FTIR portfolio

• Targets a wide range of markets, in particular, Chemicals QA/QC, Pharma

QA/QC and Academic teaching

Innovative: Sampling Flexibility Solving problems through innovation…

630 Engine DialPath TumblIR

Diamond ATR Diffuse Reflectance Transmission

• DialPath detereminacions rápides per anañisi qualitatiu o quantitatiu de

mostres líquides en varies concentraccions. Facilitat de us com ATR

• TumblIR , ideal per mostres viscoses, olis, fuels . Requereix molt poca

quantitat de mostra..

• Diamond ATR – es el accesori amb mes demanda, per la seva facilitat

d’ùs permet mesures reproduïbles de liquids, solids, gels I pastes .

• Diffuse reflectance per materials refelctants, mesura quantitativa de

particules finament dividides que dispersen com el pols

• Transmission compartment mode tradicional de mesura de líquids,

acacpta pastilles de KBr

Cary 630 Innovative Sampling Technology

INNOVATIVE: Revolutionary technology for Liquids

Analysis using the “DialPath”

Pathlength (μm) Typical Conc. Range 30 Neat - 0.1% 50 50% - 500 ppm

100 20% - 100 ppm 200 10 % - 50 ppm

TRADITIONAL APPROACH

INTUITIVE: A better approach to software… Method Driven and Guided Sampling

Software guides the

user through the

selected method.

Also recognizes the

correct sampling

interface to guide

proper sampling

techniques and

cleaning.

INTUITIVE: A better approach to software… Visual Results

Library results are shown with

the hit list and can be overlaid

or stacked

Quant results can be programmed

for critical action levels with color

coded queues (red, green, yellow)

Still the leader

in Xenon flash lamp

technology after 12 years!

Mercats i Aplicacions FTIR

Exemple 1: Anàlisi qualitatiu copolimers

Market: Bulk, Specialty and Fine Chemicals

Relative concentration of co-polymers important for

final product qualification

Poly vinyl acetate (VA) in Ethylene Vinyl Acetate

(EVA)/ Polyethylene (PE)

Standards measured, 0 – 40% VA

Ratio VA (1246 cm-1) to PE (1467 cm-1)

Linear calibration

Build Microlab PC method around calibration

Pot aplicar polimers, industria automobil, materials

electrónics , semiconductors, nanotecnologia…

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37

13

72

12

36

10

20

29

21

28

52

3500 3000 2500 2000 1500 1000

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Wavenumber

Ab

so

rbance

Polyvinyl acetate

Polyethylene

y = 5.310 (x) -0.084

1415

16

17

18

19

20

22Quant Validation Plot for Vinyl Acetate pctR²=0.999

0 1 2 3 4 5 6 7

40

35

30

25

20

15

10

5

0

Peak Area Ratio (1236cm-1 / 1467cm-1)

Vin

yl A

ce

tate

(w

t.%

)

Exemple 2: Determinació FAME en biodiesel

Biodiesel

Quantitative determination of fatty acid methyl ester

(FAME) in biodiesel blends using FTIR Spectroscopy

Exempl3 2:Seguiment de reaccions en

sintesis orgàniques.

+ Routine use

Undergrad teaching labs

Graduate research

Organic/analytical Chemistry

Identify reaction products

Teaching

Research

“Walk-up” system

Pot aplicar a estudi i caracterització de

catalitzadors heterogenis

Market: Academic +

Exemple 4: Identificació de matèries primes.

Spectra collected with 30um DialPath setting

glycerol

propylene glycol

dipropylene glycol

triacetin

Confirmació d’identitat de un producte de cosmetica abans de

consum humà. Control de Qualitat QC/QA

Pot aplicar de la mateixa manera a un fármac, aliments…

Exemple 5: Estudis de patologies (cancer, alzeimer)

amb microscopia imatge

Pot aplicar :

• Components subcelulars; identificació de compostos biologics orgánicos e inorgánicos mitjançant búsquedes en librerías espectrals.

• Estudi de processos biológics a tiemps real Optimizació de procesos de fermentació.

• Estudi de sistemes biológico en condicions ambientals específiques per analizar el plegament de enzims, dinàmica de proteínas, absorció de fármacs.

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