MASTER Study Guide of all Material- (Spectroscopy, Quinine Fluorescence , & the FTIR Infrared Lab)

What is the purpose of the Spectroscopy Lab (UV-Vis)?

To determine absorbance and calculate unknown concentration.

What is the ideal absorbance range for the Spectroscopy Lab?

0.2–1.0.

What key measurement is taken in the Quinine Fluorescence Lab?

Fluorescence intensity after excitation.

What wavelengths are used in the Quinine Fluorescence Lab?

Excitation at 350 nm and emission at 450 nm.

What is the key outcome of calibration curves in the Quinine Fluorescence Lab?

Intensity vs Concentration.

What is the blank material used in the FTIR Infrared Lab?

Air or empty crystal.

Which two stretches are indicated by key peaks in FTIR analysis?

1700 cm⁻¹ for C=O and 3400 cm⁻¹ for O–H.

In FTIR analysis, what is one method used to identify functional groups?

Record sample spectrum and identify from peaks.

What is the blank material for the Quinine Fluorescence Lab?

0.05 M H2SO4.

What type of cuvette is used in the Spectroscopy Lab?

Quartz cuvette.

What analysis tool is commonly used in the Spectroscopy Lab?

Beer's Law and linear calibration curve.

What is a critical factor to avoid in the Quinine Fluorescence Lab?

Photobleaching.

In FTIR, what is used to collect a background spectrum?

Prepare sample using ATR or KBr.

What is the relationship described by Beer's Law?

A = εcl, where A is absorbance.

What type of samples can be analyzed using FTIR?

Solids, films, and liquids.

What is a factor that can affect biological sample readings in the Quinine Fluorescence Lab?

Matrix effects.

What can the thickness of the sample be calculated from in FTIR?

Fringes.

What type of spectrophotometer is the Spec 20+?

Single beam.

What wavelength range does Cary 60 cover?

190–1100 nm.

Why is a quartz cuvette used in UV-Vis?

Allows UV transmission.

What does adjusting 0%T accomplish?

Corrects for dark current.

What does Beer's Law state?

Absorbance proportional to concentration.

True or False: Absorbance and %T are directly proportional.

False.

True or False: Always re-zero the instrument after changing wavelength.

True.

True or False: The Epoch 2 can read 96 samples simultaneously.

True.

True or False: Spec 20+ is double-beam.

False.

True or False: λmax gives maximum transmittance.

False.

Blank solution corrects for __.

Background light absorption.

The unit for molar absorptivity (ε) is __.

L/mol·cm.

Absorbance is plotted on the __ in a Beer's law plot.

y-axis.

A high R2 value (near 1.0) indicates __.

A good linear fit.

Wavelength accuracy for Cary 60 is better than __ nm.

±0.06 nm.

Why must you wipe cuvettes before inserting?

To remove fingerprints and dust that can scatter light.

What happens if you don't zero at each new wavelength on Spec 20+?

Measurements will be inaccurate due to changing lamp/detector sensitivity.

What is the slope of the calibration curve related to?

The molar absorptivity (ε) times path length (l).

What is the formula for LOD?

LOD = (3 x σ) / slope.

How do you determine an unknown's concentration?

Use calibration curve: solve.

What is C1V1 = C2V2?

The dilution equation.

What is the purpose of a calibration curve in spectroscopy?

To plot Absorbance (y) vs Concentration (x) and find the relationship between them.

What type of lamp is used in the Cary 60 UV-Vis?

Deuterium

What is the purpose of the monochromator?

Select the desired wavelength

Which part holds the sample in place?

Sample holder/cell compartment

Which component measures the amount of transmitted light?

Detector

Which of the following is not part of the optical path?

Pump

What is the correct equation for Beer's Law?

A = εcl

Absorbance is related to transmittance by:

A = -log(T)

If the cuvette path length increases, absorbance:

Increases

What does ε represent in Beer's Law?

Molar absorptivity

Beer's Law only holds at:

Low-to-moderate concentrations

What is plotted on the y-axis in a calibration curve?

Absorbance

What is on the x-axis in a calibration curve?

Concentration

The slope of the calibration line equals:

Detector response

If a standard has A = 0.300, and slope is 0.100, what is the concentration?

3.00 ppm

A linear calibration curve ensures:

Accurate prediction of unknowns

What type of cuvette is ideal for UV region?

Quartz

Why must cuvettes be wiped before inserting?

To remove fingerprints/oil that interfere with light

The blank solution should:

Contain only solvent

The purpose of the blank is to:

Zero out background absorbance

Standards must be prepared by:

Accurate dilution using volumetric tools

The most sensitive wavelength to detect an analyte is:

Lowest peak

What happens if you measure away from λmax?

Lower sensitivity

The Cary 60 software allows you to:

Choose method, read absorbance, export data

You must re-blank when:

Changing wavelength or solvent

%T stands for:

% transmittance

LOD stands for:

Limit of detection

LOQ means:

Limit of quantitation

A cuvette placed backwards might cause:

Scattered or inaccurate results

R-squared (R2) in calibration tells you:

Goodness of fit

If you forget to blank before measuring standards:

All absorbances will be offset/inaccurate

What type of transitions are observed in UV-Vis spectroscopy?

Electronic transitions.

What is the main light source in the Cary 60 spectrophotometer?

Halogen lamp.

In UV-Vis, what does λmax represent?

Wavelength of maximum absorbance.

What happens to absorbance if concentration increases?

Absorbance increases.

How is Beer's Law expressed?

A = εcl.

Why is it necessary to blank the spectrophotometer?

To account for absorbance by solvent and cuvette.

What material is used for cuvettes in UV-Vis?

Quartz.

What is typically plotted on the y-axis of a Beer's Law plot?

Absorbance.

What is the unit of molar absorptivity?

L/mol·cm.

If absorbance is 0.500 and molar absorptivity is 2000 L/mol·cm with a path length of 1 cm, what is the concentration?

2.5 × 10⁴ M.

Which range is suitable for optimal UV-Vis absorbance measurements?

0.2-1.0.

What is the result if a cuvette is inserted incorrectly?

Distorted or inaccurate absorbance reading.

What formula is used for solution dilutions?

C1V1 = C2V2.

Wavelengths selected for absorbance measurements must correspond to:

Analyte's peaks.

If the sample is too concentrated, what should you do?

Dilute the sample.

Slit width primarily affects:

Resolution and light intensity.

A dirty cuvette may cause:

Scattering and high absorbance.

Higher molar absorptivity indicates:

Stronger light absorption at a given concentration.

What will increasing the path length from 1 cm to 2 cm do?

Double absorbance.

What does LOQ stand for?

Limit of quantitation.

Fluorescence is caused by:

Emission of light after excitation.

What is the excitation wavelength in the quinine lab?

350 nm.

What is the emission wavelength for quinine?

450 nm.

Fluorescence intensity is plotted against:

Concentration.

A blank sample should contain:

Only 0.05 M H₂SO₄.

High PMT gain can cause:

Detector saturation.

Matrix effects are especially significant in samples like:

Urine and complex biological matrices.

Fluorescence emission is always:

Longer wavelength than excitation.

High background in fluorescence may be caused by:

Solvent fluorescence.

If excitation and emission slits are too wide, the result is:

Lower resolution and higher background.

Inner filter effect occurs at:

High sample concentrations.