Post on 21-Jul-2020
1
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?
29
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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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.
.