ANFISKO I-4

Aspek Kualitatif Spektrofotometer UV-Vis Data spektra UV – vis tersendiri tidak

dpt digabung u/ identifikasi Data yang diperoleh dari

spektrofotometer uv-vis sebagai parameter analisis kualitatif : adalah panjang gelombang maksimum, bentuk spektrum dan konstanta absorptivitas.

Aspek Kuantitatif

•• These methods require two measurements: These methods require two measurements:

PP00 – – the power of the radiation beam before it passes through the the power of the radiation beam before it passes through the sample sample

P –P –the power of the radiation beam after it passes through the the power of the radiation beam after it passes through the sample medium containing the analyte. sample medium containing the analyte.

•• There are two terms commonly used with absorption methods to There are two terms commonly used with absorption methods to describe the relationship of Pdescribe the relationship of P00 and P. and P.

1. 1. Transmittance Transmittance

2.2. Absorbance Absorbance

B. Absorption Methods

1. Transmittance -The fraction of incident radiation transmitted through the sample medium.

•• Major limitation – Major limitation – transmittance has a nonlinear transmittance has a nonlinear relationship with concentration of analyte in the relationship with concentration of analyte in the sample. sample.

Commonly expressed as a percentage: Commonly expressed as a percentage:

%T = T x 100%T = T x 100

I. Measurement of Transmittance and Absorbance

A.A. Transmittance (T)Transmittance (T)

WhereWhere:: PP00 = Power of incident = Power of incident beam beam

P = Power of emergent P = Power of emergent beam beam PowerPower – – Energy (in ergs) of radiation Energy (in ergs) of radiation

impinging on a 1 cmimpinging on a 1 cm22 area area of the detector per second.of the detector per second.

•• In reality, we do not measure the true PIn reality, we do not measure the true P00. We measure P. We measure PSolventSolvent, the , the power of the beam passing through a cell containing solvent only.power of the beam passing through a cell containing solvent only.

So:So:

100T%T and 0P

PT

Solvent

Solution

P

PT

B. Absorption Methods

2. Absorbance - A measurement of the amount of radiant power absorbed by the sample defined as the negative log of transmittance.

•• Absorbance has a linear relationship with sample concentration Absorbance has a linear relationship with sample concentration defined by defined by Beer’s LawBeer’s Law. .

A = A = bcbc = molar absorptivity= molar absorptivitybb = sample path length = sample path lengthcc = concentration = concentration

A = -A = -lloogg

11

00

TT

B.B. Absorbance (A)Absorbance (A)

•• The amount of radiation absorbed is proportional to the thickness of The amount of radiation absorbed is proportional to the thickness of the absorbing layer (the absorbing layer (bb), the molecular concentration (), the molecular concentration (cc) of the ) of the analyte, and the analyte, and the molecular absorbing coefficientmolecular absorbing coefficient ( (aa) of the species, ) of the species, which is characteristic of the species at a given wavelength.which is characteristic of the species at a given wavelength.

TP

P

P

PTA

Solution

Solvent

Solvent

Solution 1loglogloglog

C.C. Beer’s LawBeer’s Law

abcA •• When concentration (When concentration (cc) is expressed in molarity (mol/L) and ) is expressed in molarity (mol/L) and

thickness or pathlength (thickness or pathlength (bb) is expressed in centimeters, the ) is expressed in centimeters, the molecular absorbing coefficientmolecular absorbing coefficient ( (aa) is called ) is called molar extinction molar extinction coefficientcoefficient ( () and has units of liters per moles per centimeter [ ) and has units of liters per moles per centimeter [ L/(mol•cm) ].L/(mol•cm) ].

bcA

•• According to Beer’s Law absorbance (According to Beer’s Law absorbance (AA) is linear with respect to ) is linear with respect to pathlength (pathlength (bb) and concentration () and concentration (cc), yet there are some real ), yet there are some real limitations to this relationship.limitations to this relationship.

•• Limitations of Limitations of Beer’s LawBeer’s Law

1.1. AA has no limitation with respect to has no limitation with respect to bb. .

•• For mixtures Beer’s Law is additive. For mixtures Beer’s Law is additive.

nnnTotal

nTotal

cbcbcbcbA

AAAAA

......

......

333222111

321

or

•• Use short pathlength cells for samples of high concentration Use short pathlength cells for samples of high concentration (absorbance). (absorbance).

•• Use long pathlength cells for samples of low concentration Use long pathlength cells for samples of low concentration (absorbance). (absorbance).

Example:Example: If If AA = 0.410 in a 1.0 cm cuvette ( = 0.410 in a 1.0 cm cuvette (bb = 1.0 cm) = 1.0 cm)

Then if:Then if: bb = 2.0 cm, = 2.0 cm, AA = 0.820 = 0.820

bb = 0.1 cm, = 0.1 cm, AA = 0.041 = 0.041

Con`t Con`t Limitations of Limitations of Beer’s LawBeer’s Law

2.2. Chemical DeviationsChemical Deviations

•• AA is linear with respect to concentration ( is linear with respect to concentration (cc) except under ) except under conditions of high concentration or when chemical reactions are conditions of high concentration or when chemical reactions are occurring.occurring.

•• Above concentrations of 0.10 M the distance between analyte Above concentrations of 0.10 M the distance between analyte molecules decreases to the extent that they change each molecules decreases to the extent that they change each others charge distribution effectively changing the way they others charge distribution effectively changing the way they absorb radiation absorb radiation

(i.e. (i.e. changes). changes).

a.a. AA usually becomes nonlinear with respect to concentration when usually becomes nonlinear with respect to concentration when

cc > 0.10 M > 0.10 M

•• The same effect is observed if the salt concentration in the The same effect is observed if the salt concentration in the solvent (or buffer) is too high. solvent (or buffer) is too high.

Con`t Con`t Limitations of Limitations of Beer’s LawBeer’s Law

2.2. Chemical DeviationsChemical Deviations

•• AA is linear with respect to concentration ( is linear with respect to concentration (cc) except under ) except under conditions of high concentration or when chemical reactions are conditions of high concentration or when chemical reactions are occurring.occurring.

•• If the analyte associates, dissociates, or reacts with the If the analyte associates, dissociates, or reacts with the solvent or other components in the solution, deviations from solvent or other components in the solution, deviations from Beer’s Law will occur. Beer’s Law will occur.

b.b. AA becomes nonlinear when chemical reactions occur. becomes nonlinear when chemical reactions occur.

Example:Example:

-InHIn 2Color1 Color

H

3.3. Instrument DeviationsInstrument Deviations

•• Ideally, a monochromator will pass radiation of a single Ideally, a monochromator will pass radiation of a single wavelength, but in reality the monochromator passes a band of wavelength, but in reality the monochromator passes a band of radiation. The bandwidth of the spectrometer will affect the radiation. The bandwidth of the spectrometer will affect the linearity of Beer’s Law.linearity of Beer’s Law.

a.a. Effect of Polychromatic RadiationEffect of Polychromatic Radiation

•• Measuring absorption off the wavelength of maximum absorption Measuring absorption off the wavelength of maximum absorption ((maxmax) can affect the linearity of the Beer’s Law because of the ) can affect the linearity of the Beer’s Law because of the change in change in wrt wavelength. wrt wavelength.

•• will vary across the bandwidth, so a plot of will vary across the bandwidth, so a plot of AA vs. vs. cc can deviate can deviate from linearity.from linearity.

•• Beer’s Law will be linear when the bandwidth is < 1/10 the width Beer’s Law will be linear when the bandwidth is < 1/10 the width of the absorption peak at half height. of the absorption peak at half height.

3.3. Instrument DeviationsInstrument Deviations

•• Scattered light that reaches the detector will increase power Scattered light that reaches the detector will increase power reaching the detector. reaching the detector.

b.b. Scattered LightScattered Light

•• Long pathlength cells cause more scattered light, which causes Long pathlength cells cause more scattered light, which causes deviations from linearity in the relationship between deviations from linearity in the relationship between AA and and bb..

** Note that all instrument deviations lead to negative absorbance errors. Note that all instrument deviations lead to negative absorbance errors.

II. Instrumentation

A.A. Four Basic Types of UV-Vis InstrumentsFour Basic Types of UV-Vis Instruments•• Single-beamSingle-beam•• Double-beam in spaceDouble-beam in space•• Double-beam in timeDouble-beam in time•• Multi-channelMulti-channel

1.1. Single-beam InstrumentSingle-beam Instrument•• These can be either single channel or scanning instruments. These can be either single channel or scanning instruments.

•• 0% T is set with shutter in the beam path. 0% T is set with shutter in the beam path.

•• 100% T is set with a reference in the beam path. 100% T is set with a reference in the beam path.

•• Measurement is then made with the sample in the beam path. Measurement is then made with the sample in the beam path.

2.2. Double-beam in space instrumentDouble-beam in space instrument

•• Sample and reference are measured simultaneously and the Sample and reference are measured simultaneously and the signal from the reference is subtracted from the sample signal.signal from the reference is subtracted from the sample signal.

•• A major drawback of this type of instrument is the requirement of A major drawback of this type of instrument is the requirement of two detectors, which makes the instrument more expensive. two detectors, which makes the instrument more expensive.

3.3. Double-beam in time instrumentDouble-beam in time instrument

•• Most common type of double-beam instrument commercially Most common type of double-beam instrument commercially available.available.

•• Advantages of a double-beam over a single-beam instrument: Advantages of a double-beam over a single-beam instrument:

a)a) Compensates for variations in the source intensity.Compensates for variations in the source intensity.

b)b) Compensates for drift in the detector and amplifier (DB in Compensates for drift in the detector and amplifier (DB in time only). time only).

c)c) Compensates for variation in intensity as a function of Compensates for variation in intensity as a function of wavelength.wavelength.

4.4. Multi-channel instrumentMulti-channel instrument

•• Able to “scan” (collect) an entire spectrum in ~ 0.1 sec. Able to “scan” (collect) an entire spectrum in ~ 0.1 sec.

•• Uses signal averaging over a period of 1 sec or more to enhance Uses signal averaging over a period of 1 sec or more to enhance signal-to noise ratio. signal-to noise ratio.

•• Have high throughput of radiant energy due to the minimal optics.Have high throughput of radiant energy due to the minimal optics.

•• Typically use a deuterium lamp source for a spectral range of 200 Typically use a deuterium lamp source for a spectral range of 200 nm nm 820 nm and have a spectral bandwidth (resolution) of 2 nm. 820 nm and have a spectral bandwidth (resolution) of 2 nm.

B.B. Components of UV-Vis SpectrometersComponents of UV-Vis Spectrometers

1.1. SourceSource

a.a. Tungsten LampTungsten Lamp

•• Used for the visible region of the spectrum (350 – 800 nm). Used for the visible region of the spectrum (350 – 800 nm).

•• Usually constructed of a quartz or glass bulb containing a Usually constructed of a quartz or glass bulb containing a tungsten filament. tungsten filament.

b.b. HH22 or D or D22 lamp lamp

•• Used for the ultraviolet region of the spectrum (160 – 350 nm). Used for the ultraviolet region of the spectrum (160 – 350 nm).

•• The excited DThe excited D22 will dissociate to give a continuous band of will dissociate to give a continuous band of radiation. radiation. •• Constructed of a quartz bulb filled with deuterium and a pair of Constructed of a quartz bulb filled with deuterium and a pair of electrodes.electrodes.

c.c. Xe lamp Xe lamp

•• Used when a high intensity lamp is required and emits a Used when a high intensity lamp is required and emits a continuous band of radiation from 200 continuous band of radiation from 200 1000 nm. 1000 nm.

•• Not very common because they are expensive and have a Not very common because they are expensive and have a short lifetime. short lifetime.

B.B. Components of UV-Vis SpectrometersComponents of UV-Vis Spectrometers

2.2. Sample containers are usually cuvettes.Sample containers are usually cuvettes. •• Constructed of either quartz or glass.Constructed of either quartz or glass.

-- Glass is used for the visible region because it is transparent Glass is used for the visible region because it is transparent from 350 from 350 2000 nm. 2000 nm.

-- Quartz (silica) is used for the UV region because it is Quartz (silica) is used for the UV region because it is transparent below 350 nm. transparent below 350 nm.

•• Cuvettes are commercially Cuvettes are commercially available in pathlengths of 0.1 available in pathlengths of 0.1 10 cm. 10 cm.

3.3. DetectorsDetectors

•• Use either a photomultiplier tube or a diode array. Use either a photomultiplier tube or a diode array.

B.B. Some Typical InstrumentsSome Typical Instruments

•• Two major classifications of instruments: Two major classifications of instruments:

1.1. PhotometersPhotometers - - Simple instruments that Simple instruments that use use filtersfilters to select wavelength. They can only to select wavelength. They can only detect a single wavelength at a time, have a high detect a single wavelength at a time, have a high throughput energy due to the simple optics (good throughput energy due to the simple optics (good S/N), and are inexpensive.S/N), and are inexpensive.

2.2. SpectrophotometersSpectrophotometers - - Instruments Instruments that contain a that contain a monochromatormonochromator or or dispersive elementdispersive element that allow them to scan that allow them to scan various wavelengths. More expensive than various wavelengths. More expensive than photometers and usually have a lower S/N photometers and usually have a lower S/N due to the more complex optics.due to the more complex optics.

1.1. PhotometersPhotometers

a.a. Visible PhotometersVisible Photometers

1.1. PhotometersPhotometers

b.b. UV PhotometersUV Photometers

•• Common detector used for high performance liquid Common detector used for high performance liquid chromatography. chromatography.

•• Usually contain a Hg lamp as a source and the emission line at Usually contain a Hg lamp as a source and the emission line at 254 nm is isolated by a filter. 254 nm is isolated by a filter.

•• Good for the detection of organic Good for the detection of organic molecules, which absorb at 254 molecules, which absorb at 254 nm.nm.

c.c. Probe Type PhotometersProbe Type Photometers

•• Contain fiber optics and a mirror to Contain fiber optics and a mirror to project the source beam through project the source beam through the sample.the sample.

•• Commonly found in field Commonly found in field instruments and eliminate the need instruments and eliminate the need for a sample cell.for a sample cell.

2.2. SpectroSpectrophotometersphotometers

a.a. Visible SpectrophotometersVisible Spectrophotometers

•• Inexpensive ($500 to $3000) Inexpensive ($500 to $3000)

•• Range: 380 – 800 nm Range: 380 – 800 nm

•• Resolution: 8 –20 nm Resolution: 8 –20 nm

2.2. SpectroSpectrophotometersphotometersbb.. Single-beam and Double-beam UV-Single-beam and Double-beam UV-Vis Vis

SpectrophotometersSpectrophotometers•• Commonly use exchangeable tungsten and DCommonly use exchangeable tungsten and D22 lamps. lamps.

•• Range: 200 – 900 nmRange: 200 – 900 nm

•• Cost: $3000 - $8000 for SB, $4000 - $15,000 for DB Cost: $3000 - $8000 for SB, $4000 - $15,000 for DB

•• Resolution: 0.5 to 8 nm for SB, 0.1 to 3 nm for DB Resolution: 0.5 to 8 nm for SB, 0.1 to 3 nm for DB

•• Accuracy: ±0.005 A for SB, ±0.003 A for DBAccuracy: ±0.005 A for SB, ±0.003 A for DB

2.2. SpectroSpectrophotometersphotometersc.c. Double-dispersing UV-VisDouble-dispersing UV-Vis Spectrophotometers Spectrophotometers

•• Light passes through the monochromator twice resulting in very Light passes through the monochromator twice resulting in very high resolution.high resolution.

•• Expensive: > $10,000 Expensive: > $10,000

•• Bandwidth: 0.07 nm Bandwidth: 0.07 nm

•• Stray light: 0.0008% Stray light: 0.0008%

2.2. SpectroSpectrophotometersphotometersd.d. Diode Array UV-VisDiode Array UV-Vis Spectrophotometers Spectrophotometers

•• Can be very compact in size. Can be very compact in size.

•• Fast scanning (~ 0.1 s) Fast scanning (~ 0.1 s)

•• Range: 200 Range: 200 820 nm 820 nm

•• Bandwidth: 2 nm Bandwidth: 2 nm

•• Cost: $3000 Cost: $3000 $20,000 $20,000