Transcript Slide 1

‫بنام خدا‬
‫عباس بهرامی‬
‫عضو هیات علمی گروه بهداشت حرفه ای‬
‫دانشکده بهداشت‬
‫دانشگاه علوم پزشکی کاشان‬
‫‪[email protected]‬‬
PHOSPHINE
6002
PH3 MW: 34.00 CAS: 7803-51-2
RTECS: SY7525000
METHOD: 6002, Issue 2 EVALUATION:
FULL Issue 1: 15 August 1994
Issue 2: 15 January 1998
OSHA : 0.3 ppm
NIOSH: 0.3 ppm; 1 ppm STEL
ACGIH: 0.3 ppm; 1 ppm STEL
(1 ppm = 1.39 mg/m3 @ NTP)
PROPERTIES: gas, BP 87.8 C;
vapor density 1.17
(air = 1); spontaneously flammable
in air if
P2H4 is present
SYNONYMS:
hydrogen phosphide;
phosphorous hydride;
phosphorated
hydrogen;
phosphorous
trihydride
SORBENT TUBE
(Hg(CN)2-coated silica gel, 300 mg/150
mg)
FLOW RATE: 0.01 - 0.2 L/min
VOL-MIN: 1 L @ 0.3 ppm
-MAX: 16 L
SHIPMENT: routine
SAMPLE
STABILITY: 7 days @ 25 C
BLANKS: 2 to 10 field blanks per set
SAMPLING
RANGE STUDIED: 0.195 to 0.877
mg/m3 [1]
(16-L samples)
BIAS: 0.4%
OVERALL PRECISION ( rT): 0.091 @
2.64 to 17.41 μg
per sample [2]
ACCURACY: ± 17.6%
ACCURACY
TECHNIQUE: UV-VIS SPECTROMETER
ANALYTE: phosphate
EXTRACTION: 10 mL hot (65-70 C) acidic
permanganatereagent solution
DETECTOR: UV @ 625 nm
CALIBRATION: standard solutions of potassium
dihydrogen phosphate (KH2PO4)
(1.00 mL = 49.94 μg PH3)
RANGE: 0.3 to 10 μg per sample [2]
ESTIMATED LOD: 0.1 μg per sample [1]
PRECISION ( r): 0.074 @ 2.6 to 17.4 μg per
sample [2]
MEASUREMENT
APPLICABILITY: The working range is
0.013 to 0.6 ppm (0.02 to 0.9 mg/m3) for a
16-L air sample. The sampler is not
commercially available.
INTERFERENCES: The colorimetric
determination of phosphate is subject to
interference by any species that also forms a
molybdate complex
under these condition; possible interfering
species include PCl3 and PCl5 vapors and
organic phosphorous compounds
OTHER METHODS: This revises Method
S332 [2]. OSHA Method ID-180, "Phosphine
in Workplace Atmospheres", [3] employing
potassium
hydroxide-impregnated carbon media may
be used as an alternative method.
REAGENTS:
1. Potassium dihydrogen phosphate,
anhydrous,
KH2PO4, ACS reagent grade
2. Sulfuric acid, concentrated, ACS reagent
grade
3. Ammonium molybdate,(NH4)6Mo7O24·4H2O
4. Ferrous ammonium sulfate, Fe(NH4)2(SO4)2
5. Potassium permanganate, KMnO4
6. Stannous chloride, SnCl2
7. Glycerol
8. Toluene
9. Isobutanol
10 .Methanol
11. Water, deionized or distilled.
12. Mercuric cyanide, Hg(CN)2
*
13. Standard phosphate solution. Dissolve 200
mg KH2PO4 in 1 L of distilled water. (1.00 mL
= 49.94 μg PH3)
14. Molybdate solution. Dissolve 49.4 g
(NH4)6Mo7O24·4H2O and 112 mL concentrated
H2SO4 in distilled water to a total volume of 1
L.
15. Alcoholic sulfuric acid solution. Add 50 mL of
concentrated H2SO4 to 950 mL methanol.
16. Toluene-isobutanol solvent. Mix equal
volumes of toluene and isobutanol.
17. Ferrous solution. Dissolve 7.9 g
Fe(NH4)2(SO4)2 and 1 mL concentrated H2SO4
in water to a total volume of 100 mL.
18. Stannous chloride solution. Dissolve 0.4 g
SnCl2 in 50 mL glycerol (heat to dissolve).
19. Acidic permanganate solution. Dissolve
0.316 g KMnO4 and 6 mL concentrated
H2SO4 in 1 L water.
20. Mercuric cyanide solution.* Dissolve 2 g
Hg(CN)2 in 100 mL water.
*See Special Precautions
EQUIPMENT:
1. Sampler: Glass tube 12-cm long, 6-mm
O.D., 4-mm I.D., flame-sealed ends with
plastic caps, with two sections of mercuric
cyanide-treated silica gel (45/60 mesh), (front
= 300 mg, back = 150 mg), separated and
retained by silylated glass wool plugs. (See
Appendix)
2. Personal sampling pump, 0.01 to 0.2
mL/min, with flexible polyethylene or PTFE
tubing.
3. Spectrometer capable of measuring
absorbance or transmittance at 625 nm.
4. Two matched 5-cm absorbance cells, silica,
with tight fitting caps
Separatory funnel, 125-mL.
6. Beakers, 50-mL.
7. Pipets, 0.2-, 10-, and 25-mL, and
other
convenient sizes to make standard
dilutions.
8. Volumetric flasks, 10-, 25-, 100-, and
1000mL.
9. Water bath (maintained at 65-70C).
10. Graduated cylinders, g
11. Syringes, 0.5and 1.0-mL.
12. Balance.
13. Thermometer.
14. Stopwatch.
15. Barometer
SPECIAL PRECAUTIONS: Caution
should be exercised when
preparing the sampling media
because mercuric cyanide
is toxic. Work only in a hood
SAMPLING:
1. Calibrate each personal sampling pump with a
representative sampler in line.
2. Immediately before sampling, break the ends of
the silica gel tubes to provide an opening of at least
one half the internal diameter of the tube. Attach
the silica gel tube to the sampling pump with
flexible tubing.
3. Sample at an accurately known flow rate
between 0.01 and 0.2 L/min for a total sample size
of 1 to
16 L.
4. Seal tubes with plastic (not rubber) caps.
SAMPLE PREPARATION:
5. Place front and back sorbent sections in separate 50-mL beakers.
6. Add 10 mL of acidic permanganate solution to each beaker. Place in a water bath
maintained at 65
to 70 C for 90 min.
7. Decant the acidic permanganate solution into a 10-mL volumetric flask, and dilute to
volume with
distilled water.
8. Wash the silica gel twice with 3 mL portions of distilled water and decant the contents
into another
10-mL volumetric flask containing 1 mL of ferrous solution. Dilute to volume with distilled
water.
9. Add the contents of both 10-mL volumetric flasks (extract and washings) to a 125-mL
separatory
funnel.
10. Add 7.5 mL of molybdate reagent and 25 mL of toluene-isobutanol solvent to the
funnel. Shake
funnel for 60 seconds. Let the separatory funnel stand for 60 seconds to allow the aqueou
and
nonaqueous layers to separate. Discard the lower (aqueous) layer.
11. Pipet 10 mL of the nonaqueous layer into a 25-mL volumetric flask containing 10 mL of
the
alcoholic sulfuric acid solution.
‫انتظار میرود دانشجو در پایان جلسه بتواند‪:‬‬
‫‪ -1‬انواع اسپکتروفوتومتری را بیان کند‪.‬‬
‫‪ -2‬قانون بیرالمبرت را توضیح دهد‪.‬‬
‫‪ -3‬اجزاء دستگاه اسپکتروفوتومتر را بیان کند‪.‬‬
‫‪ -4‬انواع منبع نور را در اسپکتروفوتومتر نور مرئی‪-‬‬
‫ماوراء بنفش بیان کند‪.‬‬
‫‪ -5‬دو نوع ‪ cell‬یا کووت را توضیح دهد‪.‬‬
‫‪ -6‬نحوۀ تهیۀ منحنی کالیبراسیون را شرح دهد‪.‬‬
‫‪ -7‬روش تهیۀ منحنی کالیبراسیون بطریق‬
‫‪ Standard addition‬را بیان کند‪.‬‬
Titration
• Titration
– A procedure in which one substance (titrant) is
carefully added to another (analyte) until complete
reaction has occurred.
• The quantity of titrant required for complete reaction tells how
much analyte is present.
• Volumetric Analysis
– A technique in which the volume of material needed
to react with the analyte is measured
Titration Vocabulary
• Titrant
– The substance added to the analyte in a titration
(reagent solution)
• Analyte
– The substance being analyzed
• Equivalence point
– The point in a titration at which the quantity of titrant is
exactly sufficient for stoichiometric reaction with the
analyte.
Titration Vocabulary
• End point
– The point in a titration at which there is a sudden
change in a physical property, such as indicator color,
pH, conductivity, or absorbance. Used as a measure
of the equivalence point.
• Indicator
– A compound having a physical property (usually
color) that changes abruptly near the equivalence
point of a chemical reaction.
Titration Vocabulary
• Titration error
– The difference between the observed end point and
the true equivalence point in a titration
• Blank Titration
– One in which a solution containing all reagents except
analyte is titrated. The volume of titrant needed in the
blank titration should be subtracted from the volume
needed to titrate unknown.
Titration Vocabulary
• Primary Standard
– A reagent that is pure enough and stable enough to
be used directly after weighing. Then entire mass is
considered to be pure reagent.
• Standardization
– The process whereby the concentration of a reagent
is determined by reaction with a known quantity of a
second reagent.
Titration Vocabulary
• Standard Solution
– A solution whose composition is known by virtue of
the way it was made from a reagent of known purity
or by virtue of its reaction with a known quantity of a
standard reagent.
• Direct Titration
– One in which the analyte is treated with titrant, and
the volume of titrant required for complete reaction is
measured.
Titration Vocabulary
• Back Titration
– One in which an excess of standard reagent is added
to react with analyte. Then the excess reagent is
titrated with a second reagent or with a standard
solution of analyte.
Titration Calculations
• Titration Calculations rely heavily on the
ability to perform stoichiometric
calculations.
• Examples
N aO H + H C l
N aO H
HCl
V o lu m e
u s e d (m L )
M o la rity (M )
N a O H + H 2S O 4
N aO H
H 2S O 4
10
3 4 .3
10
1 7 .1
0 .6
0 .1 7 5
0 .6
0 .1 7 5
1. Neutralization
2. NaOH + HCl  H2O + NaCl
2NaOH + H2SO4  2H2O + Na2SO4
3. Twice as much HCl was required. Because it
takes twice as much HCl (one H) as H2SO4
(two Hs) to neutralize the same amount of
NaOH
4. H2SO4(aq) + 2NaOH(aq)  2H2O + Na2SO4(aq)
# L H2SO4=
0.010 L NaOHx0.60 mol NaOH 1 mol H2SO4
L H2SO4
x
x
L NaOH 2 mol NaOH 0.175 mol H2SO
= 0.01714 L = 17.1 mL
5. #H x MA x VA = #OH x MB x VB
Titration problems
1. What volume of 0.10 mol/L NaOH is needed
to neutralize 25.0 mL of 0.15 mol/L H3PO4?
2. 25.0 mL of HCl(aq) was neutralized by 40.0
mL of 0.10 mol/L Ca(OH)2 solution. What
was the concentration of HCl?
3. A truck carrying sulfuric acid is in an accident.
A laboratory analyzes a sample of the spilled
acid and finds that 20 mL of acid is neutralized by 60 mL of 4.0 mol/L NaOH solution.
What is the concentration of the acid?
4. What volume of 1.50 mol/L H2S will neutralize a solution containing 32.0 g NaOH?
Titration problems
1. (3)(0.15 M)(0.0250 L) = (1)(0.10 M)(VB)
VB= (3)(0.15 M)(0.0250 L) / (1)(0.10 M) = 0.11 L
2. (1)(MA)(0.0250 L) = (2)(0.10 M)(0.040 L)
MA= (2)(0.10 M)(0.040 L) / (1)(0.0250 L) = 0.32 M
3. Sulfuric acid = H2SO4
(2)(MA)(0.020 L) = (1)(4.0 mol/L)(0.060 L)
MA = (1)(4.0 M)(0.060 L) / (2)(0.020 L) = 6.0 M
4. mol NaOH = 32.0 g x 1 mol/40.00 g = 0.800
(2)(1.50 mol/L)(VA) = (1)(0.800 mol)
VA= (1)(0.800 mol) / (2)(1.50 mol/L) = 0.267 L
Titration summary
• For titrations we use the formula:
#H x MA x VA = #OH x MB x VB
Or NA x VA =
NB x VB
• NA is the combination of MA and #H
• N is also known as normality
• You can think of it a neutralizing power
• We will stick with the first equation, you do not
have to know N or what it stands for
• This is a simplification of stoichiometry. We
could get the same answer by working with
moles (n = MV) and by using the balanced
chemical equation
Titration showdown
• Titration competition (best
with a burette): find the
concentration of an H2SO4
solution – it could be
anywhere from 0-18 mol/L
• You will use the NaOH that you prepared two
weeks ago and your vast knowledge of titration
procedures and formulas.
• The wining team, is the team closest to the
correct value.
• The only restriction is : don’t put base in the
burette – only acid.
•
•
•
•
•
•
•
•
Sources of error
NaOH was not exactly 0.10 mol/L
Calculation errors (e.g. converting mL to L)
Not rinsing and drying the beaker for the acid
Over titrating (ideally, 2 titrations should be
done – one to get a rough estimate, and one to
get the exact value).
Acid or base left on the side of the flask or on
the tip of the burette (for greater precision,
water is used to rinse the tip of the burette).
Errors reading volumes.
Using pipette (10 mL is measured from 0 mL to
10 mL, not from 10 mL to empty)
Using 10 mL of base vs. 25-50 mL
For more lessons, visit
www.chalkbored.com
ALKALINE DUSTS 7401
NaOH, KOH, LiOH, MW : 40.00
(NaOH); CAS: 1310-73-2 RTECS:
WB490000
(NaOH)
and basic salts 56.11 (KOH) 1310-58-3
TT2100000 (KOH)
23.95 (LiOH) 1310-65-2 OJ6307070
(LiOH)
METHOD: 7401, Issue 2
EVALUATION: FULL Issue 1: 15
February 1984
Issue 2: 15 August 1994
OSHA : 2 mg/m3 (NaOH)
NIOSH: C 2 mg/m3/15 min
(NaOH); Group I Pesticide
ACGIH: C 2 mg/m3 (NaOH)
PROPERTIES: basic,
hygroscopic, caustic solids and
aerosols; VP not significant
SYNONYMS: alkali; caustic soda; lye; sodium hydroxide;
potassium hydroxide
SAMPLING
SAMPLER: FILTER
(1-μm PTFE membrane)
FLOW RATE: 1 to 4 L/min
VOL-MIN: 70 L @ 2 mg/m3
-MAX: 1000 L
SHIPMENT: routine
SAMPLE
STABILITY: at least 7 days @ 25 °C [1,2]
BLANKS: 2 to 10 field blanks per set
PTFE Polytetrafluoroethylene;
polyperfluoroethylene; tetrafluoroethene
homopolymer; Teflon.
MEASUREMENT
TECHNIQUE: ACID-BASE TITRATION
ANALYTE: OH- (alkalinity)
EXTRACTION: 5.00 mL 0.01 N HCl, 15 min under
nitrogen with stirring
TITRATION: 0.01 N NaOH under nitrogen, endpoint
by pH electrode
CALIBRATION: 0.01 N NaOH standardized with
0.01 N HCl
RANGE: 0.14 to 1.9 mg (as NaOH) per sample [1]
ESTIMATED LOD: 0.03 mg per sample (as NaOH) [1]
(7 x 10-4 moles of alkalinity)
PRECISION (S r): 0.033 @ 0.38 to 1.5 mg NaOH
per sample [1]
ACCURACY
RANGE STUDIED: 0.76 to 3.9 mg/m3 [1]
(360-L samples)
BIAS: 5.6%
OVERALL PRECISION (Sˆ rT): 0.062 [1]
ACCURACY: ± 16.2%
APPLICABILITY: The working range is
0.4 to 5.4 mg/m 3 for a 360-L air sample.
The method measures total alkalinity of
alkali
hydroxides, carbonates, borates, silicates,
phosphates, and other basic salts,
expressed as equivalents of NaOH.
INTERFERENCES: Carbon dioxide in the air
may react with alkali on the filter to produce
carbonates but does not interfere when
titrated. The carbonates will produce the
equivalent amount of strong alkali that was
consumed on the filter [1]. Acid a erosols
may neutralize the sample, if present, producing
a negative interference.
OTHER METHODS: This revises Methods S381
[2] and P&CAM 241 [3].
NIOSH Manual of Analytical Methods (NMAM),
Fourth Edition, 8/15/94
REAGENTS:
1. Sodium carbonate, primary standard grade.
2. Hydrochloric acid stock solution, 0.1 N.
Standardize with sodium carbonate primary
standard.
3. Dilute hydrochloric acid, 0.01 N. Dilute 10.0
mL 0.1 N stock HCl to 100 mL in a volumetric
flask with distilled water.
4. Water, distilled, CO 2-free. Boil and cool under
N2 or bubble nitrogen through distilled water
for 30 min. Store with an Ascarite trap.
5. Nitrogen, compressed.
6. Sodium hydroxide, 50% w/v.* Dissolve 50 g
NaOH in CO 2-free distilled water and dilute to
100 mL.
7. Stock sodium hydroxide, 0.1 N. Dilute 8 mL
50% NaOH to 1.0 L with CO 2-free distilled
water. Store under Ascarite or other CO 2absorbing trap.
8. Working sodium hydroxide solution, 0.01 N.
Dilute 10 mL stock (0.1 N NaOH) to 100 mL
with CO2-free distilled water.
9. Standard buffer solutions, pH 4 and 7.
* See Special Precautions
EQUIPMENT:
1. Sampler: 37-mm diameter PTFE membrane
filter (Millipore, Fluoropore or equivalent), 1.0μm pore size, supported by a cellulose backup
pad in a cassette filter holder.
2. Personal sampling pump, 1 to 4 L/min, with
flexible connecting tubing.
3. pH meter with pH electrode and recorder.
4. Titration vessel, 150 to 200 mL beaker, flask
or jar with cover containing openings for the
pH electrode and N 2 inlet and outlet.
5. Stirrer, magnetic, and stir bar.
6. Glass rod, ca. 5-mm diameter and 10 cm long
to hold filter under liquid surface in titration
vessel.
7. Pipets, 5- and 10-mL.
8. Volumetric flasks, 100-mL and 1-L.
9. Burets, 50-mL, readable to 0.1 mL.
10. Tweezers.
SPECIAL
SPECIAL PRECAUTIONS: NaOH
solutions are corrosive to tissue [4].
Handle with care.
1. Calibrate each personal sampling
pump with a representative sampler in
line.
2. Sample at an accurately known flow
rate between 1 and 4 L/min for a sample
size of 70 to 1000
L. Do not exceed a filter loading of ca. 2
mg total dust.
SAMPLE PREPARATION:
3. Transfer the sample filter to a titration
vessel with tweezers. Place the filter face
down in the
titration vessel.
4. Place the end of a glass rod in the center
of the filter to maintain the filter below the
liquid
surface during the analysis.
5. Cover the titration vessel, add 5.00 mL
0.01 N HCl, start the magnetic stirrer and N
2 purge (ca.
0.1 L/min).
6. Allow to stand 15 min (with stirring).
CALIBRATION AND QUALITY CONTROL:
7. Calibrate the pH meter with pH 4 and pH 7 buffer
solutions.
8. Standardize aliquots of the 0.1 N HCl stock
solution with sodium carbonate in triplicate [3].
a. Dry 3 to 5 g primary standard grade Na 2CO3 at
250 °C for 4 h. Cool in a desiccator.
b. Weigh ca. 2.5 g Na 2CO3 to the nearest mg.
Dissolve and dilute to exactly 1 L with CO 2-free
distilled water. The concentration is ca. 0.05 N
Na2CO3.
c. Place 5.00 mL 0.05 N Na2CO3 solution into a
titration vessel and titrate potentiometrically to
a pH of 5.
NIOSH Manu
d. Remove electrodes, rinse them into the
titration vessel, and bubble N 2 gas through
contents
of the titration vessel for 3 to 5 min to
remove dissolved CO 2.
e. Proceed with the titration to the inflection
point.
f. Calculate the normality of the stock HCl
solution
‫انتظار میرود در پایان جلسه دانشجو بتواند‪:‬‬
‫‪ -1‬اساس کار اسپکتروفوتومتر را بیان کند‪.‬‬
‫‪ -2‬در مورد قانون بیر المبرت توضیح دهد‪.‬‬
‫‪ -3‬انواع اسپکترو فوتومتر را نام ببرد‬
‫‪ -3‬قسمتهای مختلف دستگاه اسپکترو فوتومتر را‬
‫توضیح دهد‪.‬‬
UV-visible spectroscopy
How They Work
Fundamentals of modern UV-visible spectroscopy
‫کاربرد اسپکترو فتو متری در بیو شیمی‬
‫‪1‬‬
‫– اسپكتروفتومتري‬
‫بوسیله اسپكتروفتومتر شدت رنگ محلولها را با دقت اندازه گیري مي كنند‪.‬‬
‫دستگاههائي كه براي رنگ سنجي به كار برده مي شوند الكتروفتومترو یا‬
‫اسپكتروفتومتر نامیده مي شوند‪.‬‬
‫شماي ساده زیر قسمتهاي مختلف یك الكتروفتومتر را نشان مي دهد‪.‬‬
What is Spectroscopy?
• The study of molecular structure and
dynamics through the absorption,
emission and scattering of light.
Fundamentals of modern UV-visible spectroscopy
The Electromagnetic
Spectrum
E = hn
Fundamentals of modern UV-visible spectroscopy
n=c/l
Spectroscopy
Spectral Distribution of Radiant Energy
Wave Number (cycles/cm)
X-Ray
UV
200nm
Visible
400nm
IR
800nm
WAVELENGTH(nm)
Fundamentals of modern UV-visible spectroscopy
Microwave
BEER LAMBERT LAW
Light
I0
I
Glass cell filled with
concentration of so lution (C)
As the cell thickness increases, the intensity of I
(transmitted intensity of light ) decreases.
Fundamentals of modern UV-visible spectroscopy
‫الف( دانسیته اپتیك یا آبسوربنس‬
‫نوري كه از یك محلول رنگي عبور مي كند مقداري از آن جذب محلول رنگي‬
‫مي شود ‪.‬‬
‫اگر ‪ Io‬شدت نور اولیه و ‪ I‬شدت نور خارج شده از محلول باشد بنابراین ‪I a‬‬
‫شدت نوري است كه جذب محلول رنگي گردیده است‪.‬‬
‫‪Io - I = Ia‬‬
‫‪Log Io - log I =OD = Absorbanc‬‬
‫‪logI/I0=OD‬‬
‫قانون بیر المبرت‬
‫‪logIo/I= KCL‬‬
‫كه در آن ‪ K‬ضریب جذب ماده مورد آزمایش‪ C ،‬غلظت جسم مورد آزمایش و‬
‫‪ L‬قطر لوله مي باشد و چون معموالً قطر لوله را مساوي یك سانتي متر‬
‫اختیار میكنند بنابراین خواهیم داشت‪:‬‬
‫‪OD= log I0/I=KC‬‬
‫‪ K‬یا ضریب جذب ماده براي مواد مختلف فرق مي كند و به آساني قابل اندازه‬
‫گیري است‪.‬‬
‫میزان عبور یا ترا نسمیتا نس‬
‫ب( ترانسمیتانس )عبور( ‪:‬‬
‫مقدار نوري است كه از محلول رنگي مورد آزمایش خارج‬
‫مي شود‪،‬‬
‫اگر شدت نور اولي ‪ I0‬و نور خارج شده ‪ I‬باشد ‪:‬‬
‫‪I0-I=Ia‬‬
‫‪OD=Log1/T‬‬
‫‪Log I0/I=Log1/T‬‬
‫‪I/I0=T‬‬
‫اندازه گیري غلظت یك محلول‬
‫به دو روش مي توان غلظت یك محلول را اندازه گیري كرد‪:‬‬
‫الف( از راه اندازه گیري نوري كه از محلول رنگي خارج مي شود و‬
‫به آن ‪،Transmitance ،‬یا عبور می گویند؛‬
‫ب( اندازه گیري مقدار نوري كه جذب محلول رنگي مي شود و به آن‬
‫دانسیته اپتیك ‪(OPTICAL DENSITY) O.D‬‬
‫گویند‪.‬‬
‫یا آبسوربنس مي‬
The Beer-BouguerLambert Law
A   log T   log  I / I 0   log  I 0 / I     b  c
Fundamentals of modern UV-visible spectroscopy
R- Transmittance
R=
I
I0
I0 - original light intensity
I- transmitted light intensity
% Transmittance = 100 x I
I0
1
Absorbance (A) or optical density (OD) = Log
T
= Log
I0
= 2 - Log%T
I
I
Log
is proportional to C (concentration of solution)
and is I0
also proportional to L (length of light path
Fundamentals of modern UV-visible spectroscopy
through the solution).
‫بطور كلي هر اسپكتروفتومتر‪:‬‬
‫•‬
‫یك منبع نوراني‪،‬‬
‫• یك وسیله ایجاد نور تكرنگ با طول موج مشخص‬
‫•‬
‫یك سلول یا لوله مخصوص براي جادادن محلول رنگي‬
‫• ‪ ،‬یك سلول فتوالكتریك و یك گالوانومتر دارد‪.‬‬
Light Sources
UV Spectrophotometer
1.
Hydrogen Gas Lamp
2.
Mercury Lamp
Visible Spectrophotometer
1.
Tungsten Lamp
InfraRed (IR) Spectrophotometer
1.
Carborundum (SIC)
Fundamentals of modern UV-visible spectroscopy
Dispersion Devices
• Non-linear dispersion
• Temperature sensitive
• Linear Dispersion
• Different orders
Fundamentals of modern UV-visible spectroscopy
Dispersion of
polychromatic light with a
prism
Infrared
monochromatic
Ray
Polychromatic
Ray
PRISM
Red
Orange
Yellow
Green
SLIT
Blue
Violet
Ultraviolet
Polychromatic Ray
Monochromatic Ray
Prism - spray out
the spectrum
and choose the certain
Fundamentals
of modern UV-visible spectroscopy
wavelength (l) that you want by moving the slit.
Photomultiplier Tube
Detector
• High sensitivity at
low light levels
• Cathode material
determines spectral
sensitivity
• Good signal/noise
• Shock sensitive
Anode
Fundamentals of modern UV-visible spectroscopy
The Photodiode Detector
• Wide dynamic range
• Very good
signal/noise at high
light levels
• Solid-state device
Fundamentals of modern UV-visible spectroscopy
Conventional
Spectrophotometer
Schematic of a conventional single-beam spectrophotometer
Fundamentals of modern UV-visible spectroscopy
Conventional
Spectrophotometer
Optical system
of a double-beam spectrophotometer
Fundamentals of modern UV-visible spectroscopy
Conventional
Spectrophotometer
Optical system of a split-beam spectrophotometer
Fundamentals of modern UV-visible spectroscopy
Cells
UV Spectrophotometer
Quartz (crystalline silica)
Visible Spectrophotometer
Glass
IR Spectrophotometer
NaCl
Fundamentals of modern UV-visible spectroscopy
Cell Types I
Open-topped rectangular standard cell (a)
Fundamentals
of modern
UV-visible
spectroscopy
and apertured
cell
(b) for
limited
sample volume
Cell Types II
Micro cell (a) for very small volumes and flow-through cell (b)
for automated applications
Fundamentals of modern UV-visible spectroscopy
SPECTROMETRIC ANALYSIS USING
STANDARD CURVE
Absorbance at 540 nm
1.2
0.8
0.4
1
2
Concentration (g/l) glucose
3
4
Avoid very high or low absorbencies when drawing a
standard curve. The best results are obtained with 0.1 < A
Fundamentals of modern UV-visible spectroscopy
< 1. Plot the Absorbance
vs. Concentration to get a
straight line
Relating Absorbance and
Transmittance
• Absorbance rises linearly with
concentration. Absorbance is
measured in units.
• Transmittance decreases in a nonlinear fashion.
• Transmittance is measured as a %.
• Absorbance = log10
– (100/% transmittance)
Fundamentals of modern UV-visible spectroscopy
Precision and Accuracy
Precision –
Precision +
Precision –
Precision +
Accuracy –
Accuracy –
Accuracy +
Accuracy +
Fundamentals of modern UV-visible spectroscopy
Thanks for
your kind
attention
Fundamentals of modern UV-visible spectroscopy