Fundamental Techniques and Measurements

Mass MeasurementsVolume MeasurementsPreparation of a solution of known

concentrationUV-Visible Spectrophotometer

600 g

Electronic Balance

What does an electronic balance measure? _____

If you took an electronic balance with a capacity of 100 g to the moon what would its range be? _____

force

Mass: Electronic Balance

Accuracy4 to 6 significant

digits Calibration

Use known massCheck weekly or

when balance is moved

gravity

Sources of error Balance must be calibrated and

maintained in same orientation in _________ field

hydroscopic chemicals: dry to constant mass first (will increase in mass rapidly as they reabsorb water on the balance!)

When preparing a solution of a given concentration it may be difficult to get the exact mass desired_________

evaporation of wet samplesreport actual!

Electronic Balance

For maximum accuracy use balance with _______ capacity possible!

Don’t forget to clean the balance if you spill any chemicals!!!!!!

Mod el Capacity Reso lutio nDI-100 100 g 0.0001 gDI-800 800 g 0.01 gDI-5000 5000 g 0.1 g

lowest

Volume

Volumetric flaskaccuracy of ______/100 mL

Graduated cylinderaccuracy of ______/100 mL

Beakeraccuracy of _____/100 mL

Pipetteaccuracy of ± _____ for 100-1000 µLaccuracy of ± _____ for 10-100 µl

0.16 mL

1 mL

5 mL

0.6%

0.8%

What will accuracy of solution be if you use pipette, volumetric flask, and electronic balance? ________1 %

What controls the accuracy? _______Pipette

Digital Pipettes

Air displacementDo not directly contact fluid volume

avoids contamination of pipetteavoids sample carryover

Require air tight connection between tip and body

Pipette Workings

pistoncylinder

Pipette tip

Pipettes: Sources of Error

Jetting Incorrect transfer technique (getting too

much sample)Contamination from previous samplesViscous fluidsHot or cold fluidsFluids with high vapor pressure

Wipe tip on container to remove droplets

Keep Pipette vertical!

Preparation of Solutions

Example: Prepare 100 mL of a 30 mM solution of methylene blue.

The molecular weight of methylene blue (C16H18N3SCl) is 319.87 g.

concentration volumeconversion

CV M

-330 x 10 mole MBL

319.87 g MBmole MB

-3100 x 10 L 0.9596 g MB=

mass

Preparation of Dilutions

Prepare 100 mL of a 300 µM solution from the 30 mM solution

Conservation of _____mass econcentratdilute MM

econcentrateconcentratdilutedilute VCVC

econcentrat

dilutediluteeconcentrat C

VCV

= 1 mL300 M 100 mL

30 mMa fa fa fVconcentrate

Preparation of Solutions

Fill volumetric flask half way with distilled water

Add reagent (could be solid or liquid)MixFill volumetric flask to the lineMixVerify that volume didn’t change (if

necessary refill to line)

UV-Visible Spectrophotometer

TheoryInstrumentSample requirementsSoftware

Light Attenuation by an Aqueous Solution

P0

dP

dx

PP0

P

kPdx

dP

xP

P

kdxp

dP

00

kxP

P

0

ln

Ck

P is light intensity (photons/s)

Theory: Light Attenuation = f(?)

For a given excitation process, a molecule absorbs only one discrete amount of energy: expect very narrow absorption lines.

Different vibrational and rotational states yield _______ absorption lines.

Exponential decay with distance

bc =log P

PA o

broad

kxP

P

0

ln

A=bc

Po - _________ light intensityP light intensity after passing through

sampleb - ______________c - ______________ - ___________ coefficient (function

of wavelength and molecule)

incident

path length (1 cm)concentrationextinction

Absorption Spectra

Absorption Spectra for Methylene BlueBroad peaksAbsorbs _____, looks ______bluered

Instrument Light PathDiode Array

Spectrograph Lens

ShutterGrating

SlitSample Cell

Source Lens

Deuterium Lamp

Absorbance Measurement Limitations

Po is a function of the _____. If absorbance is high what is P? ______Suppose A = 3, what is Po/P? _____Suppose I create samples of higher and

higher concentration. What will happen to the absorbance measurements?

lampsmall

1000

bc =log P

PA oThere is a _________ (non zero) P that can be measured by an instrument.

minimum

A _______ keep increasing!doesn’t Amax 3

Sample Requirements

Sipper cellperistaltic pump draws sample into sipper cellrequires a few mL to displace previous cell

contents

Light source detector

pumpsample

Software

Reference (single sample) subtracts absorbance of sample

cell and reference solution usually distilled water or reagent

blank Standards (multiple samples)

used to create a __________ curve

Samples (multiple samples) after sampling, standards can be

used to estimate the concentration of samples

calibration

Maximum Absorbance: P0 is measured as reference!

Max absorbance f()________________________________________________________________________________

absorbance readings that exceed this value will not be used in analysis

lamp intensitydetector sensitivitycell absorbancereference absorbanceacceptable error

Standardsyour name

general description

rinse timesample time

sample concentrations

select number of samples by moving

this control

Samples

enter sample descriptions here

select number of samplesby moving this control