CHEM 4461/5461 INSTRUMENTAL ANALYSIS Laboratory Manual ... · PDF fileCHEM 4461/5461...

CHEM 4461/5461INSTRUMENTAL ANALYSIS

Laboratory Manual&

Course Guide

Spring Semester, 2011

Department of Chemistry and Biochemistry

The University of Texas at Arlington

2

TABLE OF CONTENTS

Section Page

Course Syllabus… 3

Laboratory Schedule… 6

Laboratory Report Format and Pre-Lab Quiz Information… 7

Overview of Spectroscopy… 11

Overview of Chromatography… 18

Experiment 1: UV-Vis Spectroscopy… 23

Experiment 2: Fluorescence Spectroscopy… 27

Experiment 3: Thermogravimetric Analysis… 28

Experiment 4: Flame Atomic Absorption Spectroscopy… 30

Experiment 5: Gas Chromatography… 33

Experiment 6: High Performance Liquid Chromatography… 38

Experiment 7: Nuclear Magnetic Resonance Demonstration… 42

Experiment 8: Mass Spectrometry Demonstration… 43

CHEM 4461/5461 Course Syllabus Instrumental Analysis

3

CHEM 4461/CHEM 5461

INSTRUMENTAL ANALYSIS (Spring 2011)

LABORATORY COURSE SYLLABUS

Instructors: Richard X. Guan

CRB, 103

817-272-6086

[email protected]

- Contact relating to miscellaneous items by email preferred

Office hours: MWF 10 – 11 am

or by appointment

Text: Skoog, Holler, and Crouch, Principles of Instrumental Analysis, 6th Ed.

and the Student Laboratory Manual.

Class Schedule: CPB 215 Section 101: T, Th: 6 – 9:50 pm

Grading: Lecture course: 500 points

Lab course (400 points total):

Experiments: 240 points (8 reports x 30 pts each)

Practicum: 140 points (oral + written portion)

Attendance/Lab Technique: 20 points

Description and Goals of the Course: This course explores the fundamental basis of chemical analysis. It

is designed to give the student a solid conceptual ground to understand how a given analytical technique

works; including its limits and advantages. The emphasis is on solutions analysis and the course is

roughly divided into: (i) Basic measurements and concepts; (ii) spectroscopy; and (iii) chromatography

and mass spectrometry.

Dropping: When dropping the course, YOU are responsible to see that the proper paperwork is filed

with the Chemistry Department. Failure to do so will result in a grade of ‛F‛.

Drop for non-payment of tuition: If you are dropped from this class for non-payment of tuition, you may

secure an Enrollment Loan through the Bursar’s Office. You may not continue to attend class until your

enrollment loan is applied to outstanding tuition fees.

../../../[email protected]


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Grade Replacement: Students enrolling in this course with the intention to of replacing a previous grade

earned in the same course must declare their intention to do so at the registrar’s office by Census Date

(Sept. 7 in F2005) of the same semester in which they are enrolled.

Pass/Fail: If P or F is a grade option in this class and you intend to take this class for a pass/fail grade

instead of a letter grade, you MUST inform the instructor(s), through the necessary paperwork, before the

Census Date.

Bomb Threat Policy: In the event of a bomb threat to a specific facility, University Police will evaluate the

threat. If required, exams may be moved to an alternate location, but they will NOT be postponed. UT-

Arlington will prosecute those phoning in bomb threats to the fullest extent of the law.

Americans with Disabilities Act: The University of Texas at Arlington is on record as being committed to

both the spirit and letter of federal equal opportunity legislation; reference Public Law 93112-The

Rehabilitation Act of 1973 as amended. With the passage of new federal legislation entitled American

with Disabilities Act-(ADA), pursuant to section 504 of The Rehabilitation Act, there is renewed focus on

providing this population with the same opportunities enjoyed by all citizens.

As faculty members, we are required by law to provide ‚reasonable accommodation‛ to students with

disabilities, so as not to discriminate on the basis of that disability. Student responsibility primarily rests

with informing faculty at the beginning of the semester and in providing authorized documentation

through designated administrative channels.

Academic Dishonesty: It is the philosophy of the University of Texas at Arlington that academic

dishonesty is a completely unacceptable mode of conduct and will not be tolerated in any form. All

persons involved in academic dishonesty will be disciplined in accordance with University regulations

and procedures. Discipline may include suspension or expulsion from the University.

‚Scholastic dishonesty includes but is not limited to cheating, plagiarism, collusion, the submission for

credit of any work or materials that are attributable in whole or in part to another person, any act

designed to give unfair advantage to a student or the attempt to commit such acts.‛ (Regents’ Rules and

Regulations, Part One, Chapter VI, Section e, Subsection 3.2, Subdivision 3.22)

Mandatory Online Safety Training: Students registered for this course must complete the University’s

required ‚Lab Safety Training‛ prior to entering the lab and undertaking any activities. Students will be

notified via MavMail when their online training is available. Once notified, students should complete the

required module as soon as possible, but no later than their first lab meeting. Until all required Lab Safety

Training is completed, a student will not be given access to lab facilities, will not be able to participate in

any lab activities, and will earn a grade of zero for any uncompleted work.

1. You should have received an email from the UTA Compliance Department. Click on the link in the

email (or navigate to https://training.uta.edu for the login page)

2. Log on using your network log-on ID and password (what you use to access email). If you do not

know your NetID or need to reset your password, visit

http://oit.uta.edu/cs/accounts/student/netid/netid.html.

3. The available courses for completion will be listed. For Chemistry 1441, complete the course entitled

‘Student Lab Safety Training’

https://training.uta.edu/

http://oit.uta.edu/cs/accounts/student/netid/netid.html


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4. If you did not receive the training email and you have not already completed the training you will

need to contact the training helpline (817-272-5100) or email [email protected].

5. Students who have not completed the training by census date may be dropped from the lab (and

consequently the lecture).

Once completed, Lab Safety Training is valid for the remainder of the same academic year (i.e. through

next August) for all courses that include a lab. If a student enrolls in a lab course in a subsequent

academic year, he/she must complete the required training again.

All questions/problems with online training should be directed to the University Compliance Services

Training Helpline at 817-272-5100 or by emailing [email protected].

mailto:[email protected]

mailto:[email protected]

CHEM 4461/5461 Laboratory Schedule Instrumental Analysis

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LABORATORY SCHEDULE (Spring 2011)

Experiment 1: UV-VIS Spectrophotometry (2 lab periods)

Experiment 2: Fluorescence Spectrophotometry (2 lab periods)

Experiment 3: Thermogravimetric Analysis (2 lab periods)

Experiment 4: Atomic Absorption Spectrophotometry (2 lab periods)

Experiment 5: Gas Chromatography (2 lab periods)

Experiment 6: Liquid Chromatography (2 lab periods)

Experiment 7: NMR Spectroscopy (Demo Lab, 1 lab period)

Meet in CRB B20 at 6:00 pm

Experiment 8: Mass Spectrometry (Demo Lab, 1 lab period)

Meet in CPB 234 at 6:00 pm

Week of: Task

01/18: No Labs; Complete safety requirements (http://compliance.uta.edu/training)

03/15: No Labs ; Spring break.

03/17: No Labs ; Spring break.

Group1 Group2 Group3 Group4 Group5 Group6

02/01: Expt 1 Expt 2 Expt 3 Expt 4 Expt 5 Expt 6






Section 101

03/22:

03/22 (Tu): Expt 7

03/24 (Th): No Labs

03/29:

03/29 (Tu): Expt 8

03/31 (Th): No Labs

03/31: Presentations of Practicum Experiments; Location TBA

Practicum experiments distributed in lecture class on Thursday, February 24

04/05 – 05/05: Perform Practicum Experiments

Practicum Lab Reports due Friday, May 6 by 5:00 pm

http://compliance.uta.edu/training

CHEM 4461/5461 Lab Report Format and Pre-Lab Quizzes Instrumental Analysis

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LABORATORY REPORT FORMAT and PRE-LAB QUIZZES Each lab report + pre-lab quiz is worth 40 points

Your laboratory report for each experiment should contain the following information.

It should roughly conform to the format of a standard scientific research article.

I. Introduction (5 points)

a. Background on analytical technique

i. Theory

ii. Advantages and disadvantages relative to other techniques

iii. Common applications (several literature references appropriate here)

b. Aim of the experiment - one or two sentences describing objective of experiment

II. Experimental (4 points)

a. Description of particular instrument used

b. Experimental procedure

III. Results (8 points)

a. Include data, required calculations, figures and tables. Each Figure and Table

should be addressed/mentioned/clarified/explained in the text of the report.

b. Describe only the results of the experiments

c. Subdivide for clarity when multiple experimental goals are present

d. Include statistics to justify non-significance of data points removed

IV. Discussion (8 points)

a. Analyze and discuss the results

b. How do the results of the experiment achieve the objective described in the

introduction?

c. Were the results the same or different than expected? WHY?!

d. If poor results were obtained, explain why. A bad result is still a result. If it is

explained then it is not wrong.

e. Answer assigned questions in the course of discussion.

V. Conclusion (5 points)

a. One paragraph summarizing: a) the objective; b) the experiment; c) the results;

d) the outcome

VI. References (5 points) – minimum three primary literature references needed (not

including websites or textbooks)

Pre-lab quiz (5 points) – Pre-lab quizzes will be administered at the beginning of each

experiment and will cover the experiment that will be performed on that day. A list of possible

(but not all-inclusive) pre-lab questions is given below. Pre-lab quizzes will only be given for

Experiments 1 – 6.


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HELPFUL HINTS/CHECKLIST (These will be very helpful…):

- Use the headings for each section given in bold type above. Keep to the format. Avoid

redundancy.

- Use references/citations (ACS Style Format) throughout to show where you found

your information (books, articles, web pages). Use a consistent format. Here you can

find a good set of guidelines: http://www.lib.berkeley.edu/CHEM/acsstyle.html

- Number figures, tables, structures, equations and refer to them in the text as they are

presented and discussed.

- Figures and tables should be numbered sequentially and should have appropriate

captions. Captions for figures are placed under the figure. Captions for tables are

placed at the top of the table.

- Figures and tables are most easily created in powerpoint. Then they can be easily pasted

into the word document (best is often “Pasted Special” as a “Window Metafile”) and

properly formatted.

- Write in essay-style prose format. Make the paper flow along the outline above. Avoid use

of bullet-point lists

- Do not include personal pronouns or personal feelings

- Write as though this is the first time such a measurement is being performed and you

are trying to communicate the procedure, the merit, the results, and the overall outcome

of the experiment.

- Look at examples of research articles in the scientific literature if you have questions

about how material should be presented. Look at e.g. Analytical Chemistry or the

Journal of the American Chemical Society

SUBMISSION:

All laboratory reports must be submitted in electronic format to your TA by 5 pm on the

Friday of the week after the lab was completed. For example, if you finish your lab experiment

on Tuesday or Thursday of one week, then the lab report is due on Friday of the following

week.

Note: All lab reports received after 5 pm on the due date will be assessed a 10% penalty. For

each additional day that the report is late, an additional 10% penalty will be assessed.

http://www.lib.berkeley.edu/CHEM/acsstyle.html


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PRE-LAB QUIZ QUESTIONS:

Expt 1 (UV-Vis)

Why is hexane a good solvent for performing measurements by UV-Vis

spectrophotometry?

Give Beer’s law and define each variable.

What is the approximate wavelength range associated with ultraviolet light?

Is visible light higher in energy or lower in energy than ultraviolet light? Explain.

Experiment 1 is broken down into three parts. Briefly describe the measurement you

will perform in each part.

Draw an example of the calibration plot you will create in your experiment related to

unknown determination. Be sure to label all parts of the plot as detailed as possible.

Expt 2 (Fluorescence)

For a particular system that fluoresces, the energy associated with fluorescence

emission is always ________________ than the energy associated with the associated

absorptive excitation.

With respect to instrumental set-up why is fluorescence spectrophotometry more

sensitive for the trace measurement of a fluorescent molecule compared to UV-Vis

spectrophotometry?

What is the sample that will serve as the unknown in your laboratory experiment?

What is the role of vibrational relaxation in the fluorescence process?

Two calibration curves will be developed in your laboratory experiment for the

fluorescence measurement of quinine. Which experimental variable will you change

to create these two separate plots?

Sketch a fluorescence spectrophotometer and label each part.

Expt 3 (TGA)

What is thermogravimetric analysis?

Why nitrogen gas is used in thermogravimetric analysis?

For what two analytes will you measure thermograms?

The heating of the hydrated salts in this experiment causes the loss of waters in

characteristic increments. How do you expect the “features” displayed in the

thermogram to change as a function of heating rate?

Does the sample need to be volatile in thermogravimetric analysis?

Expt 4 (Flame Atomic Absorption)

Draw a general diagram of a flame atomic absorption instrumental setup and label all

parts.

What metals will you focus on quantifying in this experiment?

http://composite.about.com/od/glossaries/l/bldef_t5543.htm




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What is a major limitation of flame AA in terms of monitoring the concentration of a

wide range of metals in a sample?

Why do atomic absorptions appear as sharp lines rather than the broad lines that are

observed in molecular absorption spectroscopy?

Draw and label in detail a sample calibration plot for the experiment you will

perform.

Expt 5 (Gas Chromatography)

Draw a general diagram of a gas chromatograph instrument and label all parts.

What is the carrier gas that you will employ to perform your GC experiments?

What type of detector is used for your GC experiments?

What sample will you measure for determination of Kovatt’s Retention Indices?

Define what is meant by the term “dead time” (t0)?

What parameter is calculated to determine whether or not you have achieved baseline

separation of your analytes of interest?

Expt 6 (High Performance Liquid Chromatography)

In reversed phase chromatography, polar analytes are separated based on their

relative degree of _______________________.

What type of stationary phase will you employ to perform your separations?

What are the two mobile phase components that you will use to perform you

separations?

What type of detector is used on the HPLC system you will be using?

What is meant by the term “isocratic separation”?

How many analyte components will you have to separate?

Draw and label a general diagram of an HPLC instrument.

CHEM 4461/5461 Overview of Spectroscopy Instrumental Analysis

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OVERVIEW OF SPECTROSCOPY

Spectroscopy is one of the most widely used techniques in chemistry and biochemistry.

The most widely used spectrometric methods are based upon interaction of

electromagnetic radiation (EMR) and matter.

Electromagnetic radiation (e.g. light) is a form of energy with both wave and particle

properties. What can happen to the light intensity as it passes through a medium? It

slows down in media other than vacuum because its electric vector interacts with

electric fields in the medium.

Absorption and emission are two most interesting and most useful processes when

EMR interacts with matter. When a molecule absorbs a photon, the molecule gains

energy. On the other hand, when a molecule emits a photon, the molecule loses energy.

Based on quantum mechanics, the energy of a photon depends on its frequency (v):

Ephoton = hv, where

h = Planck’s constant

h = 6.63 x 10-27 erg sec or 6.63 x 10-34 Js

Postulates of Quantum theory include:

1. Atoms, ions, and molecules exist only in discrete energy states

E0 = ground state

E1, E2 , E3 ... = excited states

- Excitation can be electronic, vibrational or rotational.

- Energy levels for atoms, ions or molecules are different.

- Measuring energy levels gives means of identification – spectroscopy.

2. When an atom, ion or molecule changes energy state, it absorbs or emits energy equal

to the energy difference.

ΔEtransition = E1 - E0 = hv = hc/


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Absorption and Emission

Absorption involves transitions from ground

state to excited states. For absorption to

occur, the energy of the photon must exactly

match the energy difference between the

ground state and one of the excited states of

the absorbing species. On the other hand, emission of EMR occurs when excited

particles (atoms or molecules) return to ground state by giving up excess energy.

Atomic Absorption and Molecular Absorption

In atomic absorption, only a few well-defined frequencies are observed in the spectrum,

since for absorption of radiation to occur, the energy of the exciting photon must exactly

match the energy difference between the ground state and one of the excited states of

the absorbing species. Compared with atomic absorption, molecular absorption is more

complex because many more potential transitions exist, i.e., electronic energy level,

vibrational energy level, and rotational energy level transitions. The energy of a

molecule is made up of three components: Emolecule = Eelectronic + Evibrational + Erotational (note

that Eelectronic > Evibrational > Erotational). Eelectronic represents the electric energy of the molecule

that arises from the energy states of its several bonding electrons; Evibrational describes the

energy associated with the interatomic vibrations that are present in molecular species;

while Erotational is the energy caused by various rotational motions within a molecule. It

should be mentioned that the number of rotational states is much larger that that of

vibrational states, and the number of vibrational states is much larger than that of

electric states.

E0

E1

En

……

.

E0

E1

En

……

.

E0

E1

En

……

.

E0

E1

vibrational level

rotational level

E2

E0

E1

vibrational level

rotational level

E2

Energy Levels - Molecular Absorption Energy Levels - Atomic Absorption


13

Beer-Lambert Law (Beer’s Law)

Beer’s Law is a fundamental law governing molecular and atomic absorption

spectroscopy. Let’s consider a beam of light with an initial radiant intensity P0 pass

through a layer of solution (with a thickness of b and concentration of c). The intensity

of the light after passage through the solution is P.

According to Beer’s law, there is a linear relationship between absorbance and

concentration of the solution:

A = -log (T) = log (P0/P) = εbc, where

Transmittance (T) = P/P0, and Absorbance (A) = -log (T)

The linear relationship between absorbance and concentration of the solution is the

basis of the quantitative detection employed by molecular and atomic absorption

spectroscopy.

Concentration (c)

Abso

rban

ce (

a)

Incident light P0

hv Emerging light P

b

Absorbing Soln. (C)


14

Electromagnetic spectrum encompasses an enormous range of wavelengths and

frequencies (or energies). And thus, various spectroscopic methods are developed by

employing part of the spectrum.


15

The following figure shows different portions of the EMR spectrum and different types

of spectroscopy involve quantum states of the atom.

UV-Visible Molecular Absorption Spectrometry is the most common analytical

technique in the analytical laboratory, which involves absorption of ultraviolet and

visible radiation for quantitative purposes. Absorption commonly occurs with many

organic molecules, metals, and metal-organic complexes. In UV-Vis, absorption

involves bonding (outer valence) electrons.

Infrared Spectrometry. Energy of IR photon is insufficient to cause electronic excitation

but can cause vibrational or rotational excitation.

Fluorescence is emission of light from any excited state of a molecule. Fluorescence

and phosphorescence are alike in that excitation is brought by absorption of photons.

The difference between them is that the electronic energy transitions responsible for the

fluorescence do not involve a change in electron spin.

Atomic Spectroscopy is based on excitation of a valence electron to an excited state. In

atomic spectroscopy, a substance is decomposed into atoms by a process called


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“atomization”. The quantity of each element is measured by absorption or emission of

UV or visible radiation by the gaseous atoms. Atomic spectroscopy methods are

categorized based on the type of atomization process, e.g., flame, electrical discharge,

inductively coupled plasma, etc. The type of atomizer used in atomization process will

affect the atomization temperature.

The following diagram is a good schematic representation of underlying energy

chances involved in UV-vis, IR, and fluorescence spectrophotometric experiments.

IR

E0

E1

vis

E2

v1

v2

v3

v4

v1

v2

v3

v4

UV

IR

E0

E1

vis

E2

v1

v2

v3

v4

v1

v2

v3

v4

v1

v2

v3

v4

v1

v2

v3

v4

v1

v2

v3

v4

v1

v2

v3

v4

UV

Spectroscopy and Energy Level Diagram

Relationship between UV, vis, and IR


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Instrumentation Available in the Laboratory

FT/IR

Bruker Vector 22 FT/IR Spectrometer

UV-vis

Jasco V-550 UV-vis Spectrophotometer

Fluorescence

Horiba Jobin Yvon FluoroMax-3 spectrofluorometer

AA

Thermo Scientific - SOLAAR M Series AA Spectrometer

CHEM 4461/5461 Overview of Chromatography Instrumental Analysis

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OVERVIEW OF CHROMATOGRAPHY

PLANAR

SUPERCRITICAL FLUID

TLC PC

GAS

GSC GLC

BPC-NP GPC GFC BPC-RP

BPCLSC IEC SEC

COLUMN

LIQUID

CHROMATOGRAPHY

PLANAR

SUPERCRITICAL FLUID

TLC PC

GAS

GSC GLC

BPC-NP GPC GFC BPC-RP

BPCLSC IEC SEC

COLUMN

LIQUID

CHROMATOGRAPHY

The chromatographic process:

1. Mobile phase (m.p.) – used for transport of the sample across the stationary phase

a. GC – gas (He)

i. Inert: does not interact with sample

b. HPLC – liquid

i. Reversed phase HPLC (RP-HPLC) – water/organic mixture

ii. Provides a differential affinity from the stationary phase for the analyte

by which a dynamic partitioning effect takes place

2. Stationary phase (s.p.) – present in the column (the “heart” of the chromatograph). A

medium which provides a differential affinity for a mixture of analytes so that each

analyte interacts to a different degree and provides separation.

a. GC – polydimethylsiloxane or other nonpolar/semipolar phase

b. RP-HPLC – C-18 phase

i. Hydrophobic chains which separate analytes based on their degree of

partitioning between the polar m.p. and the hydrophobic s.p.


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A Typical Chromatogram:

Retention time (tR)

Peak areaPeak

Height

Injection

Baseline

Time

Sig

na

l

t′Rt0

Dead time marker

Retention time (tR)

Peak areaPeak

Height

Injection

Baseline

Time

Sig

na

l

Time

Sig

na

l

t′Rt0

Dead time marker

Components of chromatograph:

1. Mobile phase delivery unit

a. GC – gas cylinder

b. HPLC – pump

2. Injector

a. GC – heated injection port to vaporize sample and deliver it onto column

b. HPLC – manual injection valve with a sample loop

3. Column – stationary phase regime; where the separation takes place

a. GC – polyimide coated fused silica coated internally with s.p.

b. HPLC – 25 cm stainless steel tube packed with silica gel particles (5 µm

diameter); the s.p. (C-18) is bonded onto the silica gel particles

4. Detector – where separated “peaks” are detected

a. GC – flame ionization detector (FID) – detects separated sample components by

“burning” the analyte

i. Others: mass spectrometer, electron capture, thermal conductivity

b. HPLC – UV spectrophotometer; absorbance detection based on analyte

chromophore (reference to UV/Vis fundamentals; Expt. 1)

i. 254 nm: highest absorbance for aromatic structural units


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ii. Others: mass spectrometer, fluorescence, electrochemical

5. Data collection – computer; converts the detector signal to chromatogram output

Qualitative Analysis

- Determination of the identity of a component in a mixture is done by analyzing

standards under the same experimental conditions as the mixture of interest. The

retention time of an analyte standard will be the same as it is in the sample mixture.

1. Standard

2. Unknown

tR(theophylline) = tR(“X”), therefore, “X” is suggested to be theophylline.

tR(theophylline) = tR(“X”)

Quantitative Analysis

- The response of an analyte (the magnitude of peak height or peak area) will be directly

proportional to the concentration of the analyte under linear operating conditions. A

calibration curve (response vs. concentration) can be made in the same manner as that

performed for previous experiments. This external standard procedure can be used to

extrapolate the unknown concentration of a component in a sample mixture.

Concentration

Re

spo

nse

Standard data points

Unknown concentration

Concentration

Re

spo

nse

Standard data points

Unknown concentration


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Instrumentation Available in the Laboratory

Gas Chromatography (GC)

Shimadzu GC-17A

He carrier gas

Split/Splitless injector

Autosampler with 10 µL syringe (Manual injection capability as well)

RTX-5 column (95% methyl, 5% phenyl polysiloxane) (15 m L, 250 µm i.d, 0.25 µm df)

Flame ionization detector

Shimadzu Class VP software (Ver. 7.4)

High Performance Liquid Chromatography (HPLC)

Varian Prostar (set up for Reverse Phase mode separations)

Binary, high pressure gradient, pumping system

MeOH and Water mobile phase reservoirs

ODS Hypersil C18 column (100 mm L x 4.6 mm i.d., 5 µm dp)

Variable wavelength UV detector

Varian Star Chrom Workstation software (Ver. 6.4)

Chromatography Terms and Definitions

1. Retention time (tR) – the time an analyte spends in the column. It is measured from the

time of injection to the peak maximum (when the analyte is eluted). It is characteristic of

an analyte for a given chromatographic method.

2. Dead time (tM or t0) – the time required to elute an unretained peak. It can used to

measure the void volume or open space in the column.

3. Adjusted retention time (t′R) – retention time minus dead time. This measures the time

an analyte spends “sorbed” in the column packing. 0ttt RR

4. Retention factor (k′) – the ratio of time the analyte spends in the stationary phase (t′R) to

the time the analyte spends in the mobile phase (t0). Also known as the capacity factor.

0t

tk R


22

5. Selectivity (α) – separation factor for quantifying the difference in retention for two

analytes of interest. α should be reported as a value greater than 1.

1

2

1

2

1

2

K

K

k

k

t

t

,R

,R

6. Width at base (W) – the portion of the baseline intersected by tangents drawn to the

peak. It is equal to 4σ of a Gaussian-shaped peak and is needed to calculate theoretical

plates (N) and resolution (R).

7. Width at half height (Wh) – the peak width at half the peak height. It is used to

calculate N and R.

8. Number of theoretical plates (N) – the number of theoretical solute equilibria for an

analyte between the mobile and stationary phases during separation in a column. N,

also called the plate number, is a measure of efficiency and the “goodness” of a column.

A column with high N produces narrow peaks. 22

54516

h

R

b

R

W

t.

W

tN

9. Height equivalent to a theoretical plate (H or HETP) – the length of column required

for one equilibrium by a solute between the stationary and mobile phases. It is the

length of one theoretical plate. H = L/N. The smaller the value of H, the more efficient

the column. H is also used to measure the rate of band broadening.

10. Average linear flow velocity (u) – linear m.p. flow usually given in cm/s. It is equal to

the length of the column (L in cm) divided by the dead time (t0 in seconds). This is used

to construct van Deemter plots for column efficiency studies.

11. Partition coefficient (K) – the ratio of concentration of an analyte in the stationary phase

to the concentration of the analyte in the mobile phase. A partition coefficient is a

fundamental physical property and is characteristic of the solute and the solvent. K is

constant for a given chromatographic method.

k

mL/g

mL/g

A

AK

.p.m

.p.s

.p.m

.p.s

12. Phase ratio (β) – the volume of the mobile phase divided by the volume of the stationary

phase in a chromatographic column.

13. Resolution (R) – a quantitative measure of the degree of separation of two peaks. R =

1.5 is baseline separation. A more comprehensive measured of resolution is given by the

Master Resolution Equation (Rs). Rs shows that resolution is a product of three

independent factors (efficiency, selectivity, and capacity).

21

12

21

12 1812

,h,h

,R,R,R,R

WW

)tt(.

WW

)tt(R

2

2

1

1

4 k

kNRs

CHEM 4461/5461 Expt. 1: UV-VIS Instrumental Analysis

23

EXPERIMENT 1: UV-VIS SPECTROPHOTOMETRY

PART 1: BEER’S LAW EVALUATION

Sample Preparation

External Standard Calibration: Create the following solutions using standard variable-

volume micropipettors and plastic tips. Create a stock solution by dissolving accurately

approximately 15 mg of quinine sulfate in 15 mL of 0.5% (v/v) sulfuric acid in a glass

vial. From this stock solution, create a series (at least 12) of diluted standard solutions

ranging from 1 to 250 mg/L. Prepare 5 mL of each solution, and again dilute with 0.5%

(v/v) sulfuric acid. Obtain 5 mL of 0.5% (v/v) sulfuric acid to use as a reference blank

solution.

Obtain ~50 mL of degassed tonic water as your unknown. This sample will be

quantitatively analyzed for quinine content using the external calibration standards

prepared above, as well as by standard addition, as follows:

Standard Addition: Deposit exactly 5.000 mL of tonic water into six 15-mL centrifuge

tubes. Using your stock solution of quinine sulfate (~1000 mg/L), aliquot 0, 100, 200,

500, 600, and 800 μL of this standard into the six separate tubes. Dilute to exactly 6.000

mL total with the appropriate volume of deionized water.

Analysis

First, be sure the instrument is on and the computer is responding. The computer

should open the Jasco software automatically. Use the instructions by the machine to

start Spectrum Measurement and create a baseline. Check with the TA if these items

are unclear. Now, in the Spectrum Measurement window, select Measurement from

the menu bar. Select parameters. Set the measurement range to 220 -450 nm, the data

pitch to 0.2 nm, and the scanning speed to 100 nm/min.

Effect of Changes in Scanning Parameters

Fill a cuvette with 0.5% (v/v) sulfuric acid and place it in the reference cell holder in the

UV-VIS spectrophotometer. Using the solution containing ~50 mg/L of stock solution,

record and print a UV spectrum from 220 – 450 nm using the parameters given above.

Next, increase the scanning speed to 1000 nm/min, change the data pitch to 0.5 nm, and

record and print a second UV spectrum. Set the scanning speed back to 100 nm/min

and adjust the data pitch to 2.0 nm. Record and print a third UV spectrum from 220 –

450 nm using these parameters.


24

Calibration Plot Linearity

Record two UV spectra from 220 – 450 nm for each of the dilute standard solutions,

which were made from the stock solution. Start with the lowest concentration. First

record a set of spectra with scanning rate set to 100 nm/min and data pitch set to 0.2 nm.

For these UV spectra record the absorbance at the peak maximum and just off the peak

maximum (Make sure to note the specific wavelengths monitored). Second, record a set

of spectra with scanning rate set to 1000 nm/min and data pitch set to 0.5 nm. Record

the absorbance at the peak maximum and just off the peak maximum for each spectrum

recorded. These numbers are found using the Spectra Analysis tweezer tool. Click on

and move the red vertical line to your peaks of interest and record the wavelength and

absorbance reported at the bottom of the Analysis window. Note, only the numbers

need to be recorded. It is not necessary to print out each of the UV spectra, but be

consistent in your choice of on-peak-maximum and off-peak-maximum wavelength

selection.

PART 2: UNKNOWN DETERMINATION

Set the scanning rate to 100 nm/min and the data pitch to 0.2 nm and record and print a

UV spectrum of your unknown degassed tonic water from 220 – 450 nm. Record

specifically the absorbance of the unknown at the same wavelengths used above (on

and off peak maximum). Place a cuvette containing 0.5% (v/v) sulfuric acid in the

sample cell and record and print a UV spectrum from 220 – 450 nm. Expand the y-axis

of the recorded spectrum so that the average noise and baseline can be estimated at the

peak maximum wavelength. Be sure to record the brand name associated with your

tonic water unknown and include this information in your lab report.

Set the scanning rate to 100 nm/min and the data pitch to 0.2 nm. Next, measure in

sequence each of your six standard addition solutions, starting with your lowest

concentration of added standard, first. Record the absorbance measured on- and off-

peak-maximum, as before, for each solution.

PART 3: Ka DETERMINATION

Sample Preparation: Standard variable volume plastic-tip micropipettors can be used to

prepare these aqueous solutions. Place 120 L of the bromcresol purple stock indicator

solution in each of 6 glass vials. Add 3840 L of pH 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 buffer

solutions to each tube, respectively. Next, place 120 L of the phenol red stock indicator


25

solution in each of 6 glass vials and similarly dilute each with 3840 L of pH 4.0, 5.0, 6.0,

7.0, 8.0, and 9.0 buffer solutions, respectively.

Spectrophotometry

Set the scanning rate to 100 nm/min and the data pitch to 0.2 nm and record a set of

spectra in the visible absorption range from 400 – 700 nm for the 12 solutions. Use a

solution made from mixing 480 L of 0.01 M NaOH with 3520 L of H2O as the

reference solution. For each set of 6 solutions containing a given indicator, overlay the

spectra and print. Also, record specifically the absorbance of the solutions at the peak

maxima below and above the wavelength of the isosbestic point. Also, record the

observed color changes for the indicator solutions as a function of pH.

DATA ANALYSIS

Make plots of absorbance versus quinine concentration (mg/L) for the four sets of data

(i.e. (a) peak maximum, scan rate 100 nm/min.; (b) off-peak maximum, scan rate 100

nm/min.; (c) peak maximum, scan rate 1000 nm/min.; (d) off-peak maximum, scan rate

1000 nm/min.) acquired in the calibration plot linearity study. Examine these plots and

identify the linear ranges of the plots. Perform a linear regression on the linear data

showing a linear response and determine the slopes and intercepts for the four plots.

Also, determine the standard deviations of the slopes and intercepts, and the relative

standard deviations of the slopes.

Using the slope and intercept of the appropriate external standard calibration plots (on-

and off-peak maximum with 100nm/min scate rate and 0.2 nm data pitch), determine

the concentration of your unknown. Also, estimate the error in this value based on the

errors in the individual values used in the calculation. From the average noise and the

standard deviation of the noise, estimate the limit of detection (LOD) of quinine in 0.5%

(v/v) sulfuric acid using the slope of the calibration plot. Using this value, estimate the

dynamic range for UV detection of quinine in 0.5% (v/v) sulfuric acid.

From your standard addition data, calculate, based on your dilutions, the final standard

concentration in each of the six samples you analyzed. Construct a standard addition

plot and determine the concentration of your unknown.

Determine the isosbestic points for the two indicators using the absorption spectra

taken at various pH’s. Also, calculate the Ka’s for the two indicators.


26

DISCUSSION POINTS

Discuss the changes in the UV spectra of quinine with changes in the scanning speed

and data pitch. What explanation for the observed changes can you offer?

Discuss the four Beer’s law plots obtained. Are deviations from linearity observed and,

if so, what explanations for these deviations can you offer? Which Beer’s law plot is

most appropriate for determining the concentration of the unknown, and why?

Evaluate the method used to determine the LOD and dynamic range for quinine in 0.5%

(v/v) sulfuric acid. Is the approach described appropriate? If not, what better

approaches might be used? If you have performed the fluorescence lab experiment,

how do those method parameters (LOD, dynamic range) compare with those obtained

in this experiment? If you have not yet performed the fluorescence experiment, how do

you think the data will compare using the different spectroscopic techniques (UV vs.

fluorescence)?

Compare the results for your quantitative analysis of quinine in tonic water by external

standard and standard addition. How well do the results agree? Explain any

discrepancies. In order to have obtained a more valid comparison of the quantitative

determination methods, what should have been done? (i.e., What procedure would

need to be followed in order to determine if the results of the two methods are the same

or different?)

Why is an isosbestic point observed in the pH dependent spectra of the two indicators?

How do the observed color changes correlate with the VIS absorption spectra of the

indicators as a function of pH? How do your calculated Ka values for the two indicators

compare with the literature values?

CHEM 4461/5461 Expt. 2: FLUORESCENCE Instrumental Analysis

27

EXPERIMENT 2: FLUORESCENCE SPECTROPHOTOMETRY

First, be sure the instrument is on and the computer is responding. Check with the TA

if either of these items is unclear. Next, using the instructions provided, set the

measurement increasement to 3 nm, the slit bandpass to 1 nm and the integration time

to 0.5 s. Finally, calibrate excitation and emission monochromators before starting your

measurements (p. 31-38 of fluorescence software manual).

PROCEDURE Prepare a stock solution of quinine in 0.5% sulfuric acid at a concentration of ~100

mg/L. Prepare ~10 mL of this solution in a 15-mL centrifuge tube.

Prepare 5 mL of a 10-fold dilution of your stock solution (~10 mg/L).

With the excitation wavelength set at 250 nm, acquire a fluorescence spectrum of the

10 mg/L solution from 350 nm to 600 nm.

Repeat the above experiment with the excitation wavelength set at 350 nm.

Prepare a set of 8 calibration solutions, spanning a range of 0.1 to 10 mg/L in regular

intervals. Prepare 2 – 3 mL of each calibration standard. Develop two calibration

curves (Intensity vs. Concentration), monitoring the emission at 450 nm using two

separate excitation wavelengths: 250 nm and 350 nm, respectively. Record the

response of each solution under each condition three times.

Take 200 L of the tonic water (unknown) and dilute up to 2.0 mL with 0.5% H2SO4.

Monitor the emission intensities for the two excitation wavelengths, as above.

Repeat two more times.

Take the 10 mg/L quinine solution and add varying amounts (0.02 g to 0.10 g) of

NaCl, monitoring the fluorescence intensity at 450 nm (use 350 nm excitation here).

Make at least 5 solutions. Plot I vs. [Cl-].

REPORT Report the structure of quinine.

Discuss the influence of the excitation wavelength on the emission spectrum.

Discuss the relative sensitivity of analysis at the two excitation wavelengths.

Discuss the quenching effect of Cl-.

Report the concentration of quinine in the tonic water and estimate the uncertainty

in this value.

If you have performed the UV-Vis experiment, compare your results from this

experiment, including the various method paramters (linear range, LOD, accuracy,

precision, etc.), with those parameters obtained by UV-Vis. If you have not yet

performed the UV-Vis experiment, conjecture as to how the methods might compare.

CHEM 4461/5461 Expt. 3: TGA Instrumental Analysis

28

EXPERIMENT 3: Thermogravimetric Analysis Procedure TURN ON NITROGEN GAS! NOT DOING THIS CAN DAMAGE THE INSTRUMENT ELECTRONICS! Log onto the computer and open the TA advantage control software. Make sure the TGA icon in the bottom right of the screen is highlighted.

1. Under summary, input the appropriate sample name and comments (e.g. Copper sulfate pentahydrate @ 10 degrees per minute). Specify a data file in the instrumental folder that includes your group #, the sample name and the date of the run.

2. Under procedure, create a name for the current method e.g. 400C 10dpm. Click the editor button and delete any current segments. Double click the ramp segment type and choose a ramp speed and maximum temperature. You will not exceed 500°C on any run.

3. Save your sequence with your group name and the ramp parameters You will need to perform the following sequences to complete the experiment. Prepare and load a fresh sample for each experiment.

1. Ramp to 300°C @ 5dpm using copper sulfate pentahydrate 2. Ramp to 300°C @ 25dpm using copper sulfate pentahydrate 3. Ramp to 500°C @ 10dpm using cobalt chloride hexahydrate

BEFORE LOADING A SAMPLE, CHECK THE INTERNAL TEMPERATURE READOUT IN THE TOP-LEFT OF THE SCREEN AND ENSURE IT IS BELOW 40°C Always exercise care when handling platinum pans as they are very expensive. Using forceps, pick up the pan by the hook and place it on the loading plate (ensure the groove on the bottom of the pan is aligned with the loading tray). Press the tare button on the front of the TGA and wait for the instrument to complete the tare cycle. Remove the pan from the loading tray and place a small amount of analyte across the surface of the pan (try to make a single uniform layer). Return the pan to the loading tray making sure to align the pan with the tray.

CHEM 4461/5461 Expt. 3: TGA Instrumental Analysis

29

Before beginning a run, check the nitrogen supply and ensure gas is flowing; if you are not sure, ask your TA; the furnace generates extreme heat and without cooling gas, the instrument will be completely destroyed. When you are ready to begin your run, click the green play button in the top-left corner of the TGA display screen. As soon as a ramp segment completes for each experimental run, you may begin to your data analysis (i.e., begin the data analysis while the instrument cools).

DO NOT BEGIN ANOTHER SEQUENCE UNTIL THE FURNACE HAS COOLED COMPLETELY. DATA ANALYSIS Using the formula mass for each analyte, determine the weight percent of one molecule of H2O. Using the universal analysis software, open your data file and begin your analysis.

1. For each transition in the thermogram, determine the total and individual changes in the weight percent of your analyte using the signal change function.

2. Determine the onset temperature for each transition using the onset point function.

DISCUSSION POINTS Discuss the difference between waters of hydration and waters of crystallization. Propose a coordination sphere for each cation. Based upon your results, propose an order for the dehydration of your analyte assuming each compound contains both waters of hydration and crystallization. Discuss any variation in the thermograms taken at different ramp speeds, and with different analytes. Your lab report should include the onset point and type of water for each molecule of H2O present in the analyte crystal.

CHEM 4461/5461 Expt. 4: AAS Instrumental Analysis

30

EXPERIMENT 4: FLAME ATOMIC ABSORPTION

SPECTROPHOTOMETRY

PART 1: SAMPLE PREPARATION

All samples must be prepared in ultra-pure water. Make 8 standards solutions (~15 mL each) of

Fe in 0.10 M nitric acid (HNO3) ranging in concentration from 1.0 ppm to 100 ppm (w/w).

Assume the density of the HNO3 solution is the same as that for pure water. Make 8 standards

solutions (~15 mL each) of Cu in 0.10 M nitric acid (HNO3) ranging in concentration from 1.0

ppm to 100 ppm (w/w). These samples will be used for calibration in flame atomic absorption

(FAA) experiments. Prepare two blank solutions (no Fe or Cu) of 0.1 M HNO3. Additionally,

obtain a substantial amount (~ 100 mL) of ultra-pure water for rinsing the flow line between

FAA measurements.

Obtain unknowns for FAA determination. Be sure to note the sample codes in your lab notebook

and in your laboratory report.

PART 2: INSTRUMENT OPTIMIZATION

Refer to the software instruction manual for setting up the method. Cookbook methods are

available for both flame and graphite furnace atomic absorption measurement of different metals

in the instrument software. Make sure to use flame methods only.

PART 3: STANDARD PLOTS

Flame Atomic Absorption

Prepare a method and sequence for analyzing your standards by FAA. Each sample will need to

be sequentially placed into the aspirator as prompted by the software. For the first

determination, use a slit width of 0.1 nm. Absorbance associated with the 8 standard Fe and

solutions and the unknown will be recorded. Be sure to print the appropriate report and record

the data in your lab notebook. Next, adjust the method so that the measurements are made at a

secondary absorption wavelength for Fe. Again measure and record the absorbance associated

with the 8 standard Fe solutions. Repeat the two standard plot determinations, using primary and

secondary wavelengths, for a slit width setting of 1.0 nm. Repeat the above for your Cu samples.

REPORT

Construct calibration curves, perform linear regressions and determine the concentration of the

unknowns supplied for FAA measurements. Perform the analysis for primary and secondary


31

wavelengths and for different slit widths, as appropriate. Explain your results in terms of LOD

and sensitivity. Do these results suggest an optimal method for determining Fe and Cu

concentrations?

Draw and label a diagram for a flame atomic absorption spectrometer. Discuss the parts of the

flame and the importance of proper alignment of the flame between the lamp and detector. Why

is a hollow cathode lamp (HCL) for each element required in AAS? How does the lamp current

affect the width of the spectral lines from the HCL and why? What are other methods for

measuring trace metals other than FAA?


32

TABLE

Elements Wave length (nm) Special band pass

(nm)

Optimum working

range (g/mL)

Ag 328.1

338.3

0.5

0.5

1-5

3-12

Al 309.3

396.3

237.3

236.7

257.4

256.8

0.5

0.5

0.5

0.5

0.5

0.5

40-200

50-250

200-800

250-1000

400-1600

650-2600

Cd 228.8

326.1

0.5

0.5

0.5-2.0

250-1000

Fe 248.3

372.0

386.0

392.0

0.2

0.2

0.2

0.2

2.5-10

25-100

50-200

800-3200

Hg 253.7 0.5 100-400

K 766.5

769.5

404.4

1.0

1.0

0.5

0.5-20

0.5-60

200-800

Mg 285.2

202.6

0.5

1.0

0.1-0.4

5-20

Pb 217.0

283.3

261.4

202.2

205.3

1.0

0.5

0.5

0.5

0.5

5-20

10-40

200-800

250-1000

2000-8000

CHEM 4461/5461 Expt. 5: GC Instrumental Analysis

33

EXPERIMENT 5: Gas Chromatography

PART 1: OPTIMIZATION OF CHROMATOGRAPHY

First, make sure the instrument in on, the carrier gas is flowing, the flame ionization

detector (FID) is lit, and the computer is responding. Record the dimensions (L, i.d.), the

make and model, and the type and thickness of the stationary phase for the column

installed in the chromatograph. Check with the TA if any of these items are unclear.

Next, using the instructions provided, set the flow rate of the column to 0.80 mL/min,

the split ratio to 30:1, and the injector and detector temperatures to 250 °C.

Sample Preparation

In an autosampler vial, deposit 20 μL of carbon tetrachloride (CCl4) using the

micropipette. In a second autosampler vial, deposit 20 μL of chloroform (CHCl3). In a

third autosampler vial, deposit 20 μL of CCl4 and 20 μL of CHCl3. Finally add sufficient

volume of dichloromethane (CH2Cl2) to each tube to bring the total volumes to 1.000 mL

and cap each vial securely.

Determining Dead Time (t0)

Each student will make five manual injections of butane to determine the dead time and

to determine the reproducibility of each individual’s injection technique. Enter a

temperature program for the column, in which the oven temperature is held constant at

50 °C. This is called an isothermal separation method. Insert the syringe needle into the

outlet of a butane lighter and press the gas release button on the lighter. Do not strike

the flint. Withdraw the plunger on the syringe to between 2 and 4 μL. Prior to

injection, keep the syringe needle pointing down to avoid loss of butane from syringe.

Inject the solution and record a chromatogram. A typical manual injection in split

injection mode should take approximately 2.5 seconds (1 second to insert the needle, 0.5

seconds to inject, and 1 second to withdraw the needle). Be sure to be consistent in

timing the start of the run with the manual injection (insert syringe inject

withdraw syringe start run). Record the retention time of the peak for each repetition

and calculate the average, the standard deviation, and the relative standard deviation

for each individual’s five replicates, as well as for the entire group. This is the dead

time for the column under this method.

Effect of Changes in Temperature Program

Enter a temperature program for the column, in which the oven temperature is held

constant at 30 °C. Inject a 0.5 μL sample of the mixed solution containing both CCl4

and CHCl3 and record the chromatogram. Print the chromatogram and the peak


34

analysis report for your records. Make sure all printed chromatograms are adequately

labeled with sample and method information for future reference. Also, expand the

chromatogram on the computer and record the width at half-height (Wh) for each peak

(Note: width at base (W) may also be recorded, if necessary and if peaks are adequately

resolved).

Enter an isothermal temperature program where the oven is set to 50 °C. Inject a 0.5 μL

sample of the mixed solution containing both CCl4 and CHCl3 and record the

chromatogram. Print the chromatogram and the peak analysis report, and record Wh

for each peak.

Enter an isothermal temperature program where the oven is set to 90 °C. Inject a 0.5 μL

sample of the mixed solution containing both CCl4 and CHCl3 and record the


for each peak.

Enter a temperature program in which the oven temperature begins at 50 °C and

increases to 90 °C at a rate of 5 °C/min. This is called a temperature gradient separation

program. Inject a 0.5 μL sample of the mixed solution containing both CCl4 and CHCl3

and record the chromatogram. Print the chromatogram and the peak analysis report,

and record Wh for each peak.

Finally, record chromatograms of 0.5 μL injections of each of the two pure compounds

dissolved in CH2Cl2 using the temperature program above. Print the chromatogram and

the peak analysis report for each run, and record Wh for each peak.

Effect of Changes in Flow Rate

Adjust the column flow of the carrier gas to 1.50 mL/min. Set the oven temperature for

an isothermal run at 50 °C, make a 0.5 μL injection of the mixed solution, and record the


for each peak. Adjust the column flow of the carrier gas to 3.00 mL/min and repeat the

experiment. Print the chromatogram and the peak analysis report, and record Wh for

each peak.

PART 2: STANDARD CURVE

Sample Preparations

In four (4) autosampler vials, deposit 30, 40, 50, and 60 μL of CCl4, respectively, using

the micropipette. Next, add 20 μL of CHCl3 to each tube. Finally, add sufficient volume


35

of CH2Cl2 to each vial to bring the total volume to 1.000 mL and cap each vial securely.

These are your calibration standard samples. Prepare a sample “blank”, by filling an

autosampler vial with 1 mL of CH2Cl2. In an autosampler vial, add 20 μL of CHCl3 to

500 μL of your unknown. Add sufficient CH2Cl2 to bring the total volume to 1.000 mL

and cap securely. This is your unknown sample for quantitative determination.

Chromatography

Place the sample vials in the autosampler and create a sequence in the software for the

analysis of your samples, using the same temperature program as previously and a

carrier gas flow rate of 0.8 mL/min. Program the autosampler to sequentially inject 0.5

μL of each of the solutions (calibration standards, blank, unknown). Print the

chromatograms and the peak analysis reports for your records.

PART 3: KOVAT’S RETENTION INDICES

Using the isothermal method with column temperature of 50 °C and flow rate of 0.8

mL/min, record the chromatogram for 0.1 μL injection of a neat mixture of the C5 – C8

hydrocarbons. Print the chromatogram and the peak analysis report for your records.

DATA ANALYSIS

Part 1

Calculate the resolution (R) for the separation of CHCl3 and CCl4 in the 4 chromatograms

recorded using the various oven temperature settings (isothermal at 30, 50, and 90 °C;

and gradient temperature programming). Also, calculate the capacity factors (k’) for

CHCl3 and CCl4 and the selectivity factor (α) resulting from each method. Assume t0 for

each of the methods is equivalent to that which was determined initially from the

butane injections at 50 °C. How do the calculated values (R, k’, α ) change for

separations using the different oven temperature methods?

Calculate R between CHCl3 and CCl4 at the 1.5 mL/min and the 3.0 mL/min flow rates.

Calculate the number of theoretical plates (N) and the height equivalent to a theoretical plate

(HETP) for CHCl3 and CCl4 at each of the 3 flow rates (0.8, 1.5, and 3.0 mL/min). Using

the HETP values, calculate the A, B, and C constants in the Van Deemter equation for

each of the two compounds (Note: You will need to convert the flow rates to linear

velocities; divide by the area of the cross section of the open portion of the column).

Finally, take the first derivative of the Van Deemter equation and estimate the optimum

column flow rate.


36

Part 2

Using the chromatograms obtained from your standard solutions perform a linear

regression of the plot of area of the CCl4 peak versus percent CCl4 (external standard

method). Determine the slope and the intercept of the best-fit line to the data. Also,

determine the standard deviation of the slope and the intercept. Next, for each of the

five chromatograms of the standard samples, divide the area of the CCl4 peak by the

area of the CHCl3 peak and make a second plot of the these values versus percent CCl4

(internal standard method). Determine the slope and the intercept of the best-fit line to

the data and determine the standard deviation of the slope and the intercept. How does

the relative standard deviation of the slope of the second plot compare with the relative

standard deviation of the slope of the first plot?

Determine the concentration of the unknown by the external standard method (using

the regression equation from the first plot, determine the concentration of unknown

from the measured area) and the internal standard method (divide the area of the CCl4

peak by the area of CHCl3 from your unknown chromatogram and, using the regression

equation from the second plot, determine the concentration of unknown from the

normalized area). Estimate the error in these values from the errors in the calibration

plot. How do the absolute values determined and their uncertainty compare? From the

blank injection, estimate the average background signal and standard deviation of this

value. From the slope of the first calibration plot, estimate the limit of detection (LOD)

and the limit of quantification (LOQ) for the CCl4.

Part 3

Make a plot of the log of the adjusted retention times for the C5 to C8 hydrocarbons

versus the number of parafinnic carbon atoms x 100 (e.g. for C5, this equals 500 and so

on). Perform a linear regression and determine the slope and the intercept of the best-fit

line. Use this plot and the adjusted retention times for CHCl3 and CCl4, calculate the

Kovat’s retention index for each compound.

DISCUSSION POINTS

In your laboratory report, discuss the reasons for using temperature programming in

gas chromatography rather than simple isothermal separations. Relate this discussion

to the specific examples in this experiment.


37

How reproducible is your injection technique versus that of the group? Why is it

important to make the injection quickly and to be consistent in your technique? Would

expect the dead time to increase or decrease with an increase in oven temperature?

Discuss the Van Deemter equation and the implications of the three characteristic

constants A, B, and C. Which of these constants are particularly relevant to gas

chromatography and why?

Compare and contrast the two calibration plots. Why is the second method of

preparing a calibration plot using an internal standard considered superior in gas

chromatography? Is this borne out in your results? If you had performed the unknown

determination using manual injection, instead of an autosampler, would you expect any

substantial differences in your determinations? In determining the LOD and LOQ,

what assumptions have you made? Are these reasonable for gas chromatography with

an FID detector?

What is the purpose of determining the Kovat’s retention indices of the 2 compounds?

How can this information and method by useful for designing additional gas

chromatographic separation methods and predicting the outcome?

CHEM 4461/5461 Expt. 6: HPLC Instrumental Analysis

38

EXPERIMENT 6: High Performance Liquid Chromatography

PART 1: OPTIMIZATION OF CHROMATOGRAPHY

First, make sure the instrument in on, the UV lamp is on, and the computer is

responding. Make sure the 2 solvent reservoirs are filled with HPLC-grade methanol

and HPLC-grade water, respectively, and that each bottle is being gently sparged by

helium to remove dissolved gases in the solvent. Record the dimensions (L, i.d.), the

make and model, the type of the stationary phase, and the type of packing for the

column installed in the chromatograph. Check with the TA if any of these items are

unclear.

Sample Preparation

The stock solution contains methyl paraben, ethyl paraben, propyl paraben, and butyl

paraben, each at a concentration of 200 ppm (v/v), in 50/50 methanol:water. Make serial

dilutions of this mixture using 50/50 methanol:water to create a series of calibration

standard mixtures at 150 ppm, 100 ppm, 75 ppm, 50 ppm, and 10 ppm concentrations.

Sample volumes of 1.000 mL are sufficient. All solutions should be filtered using a

syringe filter prior to use.

Effect of Mobile Phase Composition

Using the instructions provided, set the flow rate of the column to 1.00 mL/min, the

solvent ratio of the mobile phase to 99% methanol / 1% water, and turn the pump on.

Set the analysis time to 8 minutes and the detector wavelength to 254 nm. Check all

tubing fittings for leaks. Allow at least 5 column volumes of solvent to pass through the

column before beginning the analysis. Prior to analysis, note the pressure of the system

and make sure it is stable prior to beginning the run.

Fill your syringe with the 200 ppm mixture to make a full-loop injection (fill the

injection needle with approximately 1 – 2 μL more sample than the volume of the loop).

Make sure the injector is in the “load” position, insert the syringe and fill the loop.

Leave the empty syringe in the injector as you switch the injector to the “inject” position

and start the run. You can remove the syringe after the injection is made and the run is

started. Print the chromatogram and the peak analysis report for your records. Make

sure all printed chromatograms are adequately labeled with sample and method

information for future reference.

Repeat this analysis (using the 200 ppm mixture), sequentially, for solvent compositions

of 90/10, 80/20, 70/30, and 60/40 methanol:water. The separation under each of these


39

conditions is called an isocratic separation method, because the solvent composition

does not change throughout the analytical run. Remember to allow sufficient time for

equilibration at each new mobile phase (approximately 5 column volumes) and record

the pressure prior to each run. Print the chromatograms and the peak analysis reports

for each run. Also, expand the chromatogram on the computer and record the width at

half-height (Wh) for each peak, if possible (Note: width at base (W) may also be recorded,

if necessary and if peaks are adequately resolved). (Note: You may need to increase the

run time for methods which contain a higher proportion of water in the mobile phase.)

PART 2: STANDARD CURVE

Chromatography

Pick an isocratic separation program that provides adequate retention (k’ (peak 1) > 1)

and resolution (R > 1.5) of all three sample analytes. This can be, but does not have to

be, one of the methods investigated above. Inject each of your calibration sample

mixtures (full-loop injection) under this method, starting with the lowest concentration

(10 ppm) first. Record the retention times and areas for each peak in each calibration

sample mixture analyzed. Inject your unknowns (unknown 1 and unknown 2) under

the same conditions and record the chromatograms, also noting the retention times and

areas of the peaks in your samples. Note the sample codes for your unknowns and

indicate these in your report.

PART 3: SOLVENT GRADIENT SEPARATION

Set up the instrument to perform a gradient solvent program separation method. Start

with an initial mobile phase composition of 50/50 methanol:water, a hold at that

composition for 1 min, an increase to 99/1 methanol:water in 6 minutes, and a 1 minute

hold at the final composition, as follows (between 1.0 min and 7.0 min, the instrument

will change the solvent composition, in a linear fashion, from 50/50 to 99/1

methanol:water):

Table 1: Initial solvent gradient program

Run time (min) methanol (%) water (%)

0.0 50 50

1.0 50 50

7.0 99 1

8.0 99 1


40

Inject the 200 ppm mixture and record a chromatogram. Calculate k’ for each peak, and

the resolution between each component.

Next, modify the solvent gradient to create a method for base line separation (R ≥ 1.5) of

all three components in under 5 minutes. Note, that k’ (peak 1) must be greater than 1

for the optimized method. You may change any parameter in Table 1 above.

Remember to allow sufficient time for equilibration prior to each analytical run.

DATA ANALYSIS

Part 1

Calculate the capacity factor (k’) and number of theoretical plates (N) for each analyte

under each of the different mobile phase compositions tested. Also, calculate resolution

(R) and selectivity (α) for each pair of analyte peaks in each chromatogram. How do the

calculated values change with the different instrumental settings? What is the dead

time (t0) for your experimental set-up and how was this determined?

Part 2

Using the chromatograms obtained from your standard solutions perform a linear

regression of the plot of area of each peak versus concentration (external standard method)

for each analyte. You will have three separate calibration curves. Determine the slope

and the intercept of the best-fit lines to the data for each plot. Also, determine the

standard deviation of the slope and the intercept for each plot. Determine the

concentration of each component in your unknown mixtures and estimate the error in

each of these values from the error in each calibration plot.

Part 3

Record the solvent program for each method you try, as well as k’ (peak 1), k’ (peak 3),

and R (each pair of analyte peaks) for each trial.

DISCUSSION POINTS

In your laboratory report, discuss the reasons for using gradient solvent programming

in HPLC rather than simple isocratic separations. Relate this discussion to the specific

examples in this experiment.

What is the mechanism of a reverse phase HPLC separation? Which mobile phase

component is the “strong” component and which is the “weak” component? Why are


41

these designated as such? What other types of solvents are typically employed in

reverse phase HPLC?

Discuss the effect of mobile phase composition on the separation of your analytes in

terms of retention time, capacity factor, selectivity, and resolution. Also, discuss the

change in pressure observed for each isocratic separation method. Why is it important

that the column is “equilibrated” prior to performing each analysis? How is the

pressure an indication of equilibration?

Present the structures of the analytes and logically reason the elution order of the

compounds based on the reverse phase HPLC separation methods you have employed.

What is the identity and concentration of each analyte in your unknown mixtures?

Why is a wavelength of 254 nm set for the detector? What is the “chromophore” in

your analytes that allows them to be detected at this wavelength?

For your gradient separation method, why is it important to have k’ (peak 1) > 1 and R ≥

1.5?

Tabulate all of your data for effective presentation.

CHEM 4461/5461 Expt. 7: NMR Demo Instrumental Analysis

42

EXPERIMENT 7: Nuclear Magnetic Resonance Spectroscopy

Demonstration

Address the following points when preparing your laboratory report for this

demonstration of nuclear magnetic resonance (NMR) spectroscopy. You may need to

independently search for some of this information in your textbook, in the literature,

and in prominent websites.

Briefly describe the basic measurement that is being made in proton NMR

spectroscopy.

What is a superconducting magnet? Why is it necessary/desirable to use a

superconducting magnet in high magnetic field NMR spectroscopy? What are

the dangers associated with these instruments?

Why is it necessary to spin the sample in solution phase proton NMR

spectroscopy?

Using the magnetogyric constant for a proton, calculate the approximate

magnetic field associated with a 60 MHz instrument. Do the same for a 500 MHz

instrument.

How do the energies associated with the splitting of the nuclear spin states in

NMR compare with the energies associated with molecular rotation?

Currently there are efforts underway to create a 24 T magnet. What would be

the frequency of splitting for a proton in an instrument with this magnetic field

strength?

The x-axis of both the 60 MHz and 500 MHz NMR spectra is the same, but the

resolution of the peaks in the 500 MHz instrument is much better. What factors

contribute to the improved resolution in the 500 MHz instrument?

What is the function of the “lock solvent” in high field NMR spectroscopy?

CHEM 4461/5461 Expt. 8: MS Demo Instrumental Analysis

43

EXPERIMENT 8: Mass Spectrometry Demonstration

Address the following points when preparing your laboratory report for this

demonstration of mass spectrometry and the use of specific ion sources. You may need

to independently search for some of this information in your textbook, in the literature,

and in prominent websites (e.g. www.asms.org).

PART 1: MASS SPECTROMETRY, GENERAL ASPECTS

What are the general instrumental components which compose a mass

spectrometer?

Why is a high vacuum necessary in mass spectrometer instruments?

What is the difference between high resolution and low resolution mass

analyzers? Provide examples.

PART 2: ELECTROSPRAY IONIZATION – MASS SPECTROMETRY

Briefly describe the measurement which was made in the demonstration of ESI-

MS. Include aspects of instrumentation, sample components, and the purpose of

the experiment.

Describe the electrospray process which leads to the formation of gas phase ions.

What is the molecular form of ions created in positive mode and in negative

mode ionization?

What kind of mass spectrometer/mass analyzer was employed and what is the

basic theory behind its operation?

Briefly describe the applicability of ESI-MS to different sample types. This

information can also be effectively communicated by citing specific examples

from the literature.

How is ESI-MS capable of analyzing large molecules with masses higher than the

upper mass-to-charge limit of common mass analyzers?

Why is ESI referred to as a soft ionization source?

http://www.asms.org/

CHEM 4461/5461 Expt. 8: MS Demo Instrumental Analysis

44

PART 3: MATRIX-ASSISTED LASER DESORPTION/IONIZATION - MS

Briefly describe the measurement which was made in the demonstration of

MALDI-MS. Include aspects of instrumentation, sample components, and the

purpose of the experiment.

What is the purpose of the matrix? What processes occur to result in the

formation of ions from the analytes of interest?

Why is MALDI ideally suited to time-of-flight mass analysis detection? What are

the analytical advantages of a TOF mass analyzer?

Briefly describe the applicability of MALDI-MS to different sample types.

What is post-source decay and how is it useful?

Is MALDI a soft or hard ionization technique and why? In this respect, how does

it compare to laser desorption/ionization with no matrix?

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