1

Chem 241 - Summer 2010
Chemistry 241 About Me

Professor: Gary L. Glish
B.A. 1976 Wabash College
Office: Caudill 320
» majors: Chemistry and Economics
Phone: 962-2303
email: chem241glish@unc.edu
Ph.D. 1980 Purdue University
Office Hours: 1980-1992 Oak Ridge National
» Monday 2:00-3:00; Thursday 11:00–12:00
Laboratory
» Other afternoons - drop-in or by appointment 1992 UNC
» Teach: Chem 070, 101, 241, 241L,
395, 396, 448, 742, 742L, 743, 941,
992, 993, 994
Chemistry 241 Prerequisites Class logistics

Chem 102 or 102H blackboard.unc.edu
»Syllabus
»Lecture Notes
It is an Honor Code violation to enroll »Exam keys
in a course for which you have not
taken prerequisites.
»Old exams
»Equation sheet
Class logistics Class logistics – Schedule

Book: Quantitative Chemical Week of Monday Tuesday Wednesday Thursday Friday
Analysis, 7th Edition, by Daniel C. May 10 Chpts. 3, 4,5 Chpt. 23,24 Chpt. 24,25 No Class
May 17 Chpt. 25,26 Chpt. 9 Chpt. 10 No Class

Harris (UNC Custom text is subset of Exam 1
May 24 No Class No Class No Class No Class No Class

chapters from this)
May 31 No Class Chpt. 11 Exam 2 Chpt. 18,20 No Class
» Also recommended: Solutions Manual June 7 Chpt. 20, 21 Chpt. 21, 22 Exam 3 Chpt. 14,15 No Class
Grading -
» 3 in class exams - 20% each Final Exam
» final exam - 40% 3-6 PM, Monday, June 14th
» Ask the Class (extra credit)
Section 1 1
Class logistics - Exams Honor System (http://honor.unc.edu)

Final is comprehensive  General Responsibilities.
The final will be divided into sections, a It shall be the responsibility of every student at the University of
North Carolina at Chapel Hill to:
better score on a section of the final than 1. Obey and support the enforcement of the Honor Code;
the in-class exam will replace that grade
Missed exams will use the
2 Refrain from lying
2. lying, cheating,
cheating or stealing;
3. Conduct themselves so as not to impair significantly the welfare or
corresponding section of the final as the educational opportunities of others in the University community;
grade and
Equation sheet will be provided 4. Refrain from conduct that impairs or may impair the capacity of
University and associated personnel to perform their duties, manage
Left handers – email me by Feb. 1 if you resources, protect the safety and welfare of members of the
University community, and maintain the integrity of the University.
want a left-handed desk for exams
Honor System Procedures Exam Policies

Professor reports possible violation  Cheating on an exam - automatic 0 (can NOT
be replaced)
Student attorney general decides if  NO CELL PHONES or IPODS – automatic 0
charges
g should be filed ((can NOT be replaced)
p )
Honor system notifies accused  Exam leaves room other than with me –
student – defense counsel provided automatic 0 (can NOT be replaced)
 Exam not turned in within 60 seconds after I
Honor Court Trial say STOP – automatic 0 (can be replaced)
If convicted - Sanctions
Exam Policies Class logistics - Grading scale

Replacement of grade from Final xx Grade
G
Exam not valid for grades resulting s A 1.25
from disciplinary action x  your score B  0.45
If
If you only
l take
k the
h Final
Fi l Exam,
E that
h x  class average C -0.75
grade is your grade
s  standard deviation D  -1.4
If you miss more than 1 exam AND
the Final Exam, no replacement of You must earn at least 50% of the
grades points to pass
Section 1 2
Class logistics - Alternative scale Class logistics - Equations

92 -100%  A
84 - 91 %  B H A =
2 2
[ H+ ]
2
[ H+ ] + [ H+ ] Ka1 + Ka1 Ka2

N*
No
 g*  - E
=   e kT
 go 
 =
c
n E = h
76 - 83%  C
A' y ' b  x ' b A'
 K a1 K a2 F + K a1 K w 
1/ 2 A" y "b  x " b A" 1
[ H+ ] =   [ x]  [ y]  =
  x ' b  y ' b x 'b y 'b
68 - 75%  D
K a1 + F 
x " b  y " b x "b y "b
[Base] - b  (b2 - 4ac )

1/ 2
[ H+ ] A = -logT = bc
pH = pKa + log
[Acid] x=  HA =
2a Ka + [ H + ]
The scale that gives you the highest grade Csolute

=F
areasolute

%et  %ex  %e y ...
2 2

1
2
 
et  ex 2  e y 2 ...
1
2 Ka Kb = K w
will be used Cstandard areastandard
response to y .05916 q  nF
kx , y  E = E- log Q G = - nFE
response to x n
Class logistics - Ask the Class Class logistics - Ask the Class
Participation optional – must have a  Most questions will be worth 3 points
“clicker”  Music questions – 1 point for answering
Questions asked during class  It is an Honor Code violation to answer
with someone else’s clicker
Fraction
F i off totall points
i over theh
semester multiplied by 10 and added
to final exam grade to calculate final
grade
Class logistics - Homework Course Goals

Suggested homework problems on Learn some basics about
Blackboard Analytical Chemistry
Homework will not be graded or
collected Understand pprinciples
p of
Work on homework problems with techniques
classmates Apply principles to problem
At least one question on each exam will
be a homework problem solving (THINKING)
Section 1 3
What is Analytical Chemistry? What is an “Analyte”
Analytical chemistry is object of the chemical

the science of chemical measurements,
measurements sometimes the “sample”
sample
 sometimes a component in
the “sample”.
What are we trying to measure? Define what is being measured

Qualitative Analysis - what is it?
surface composition
»Newcastle or Coors?
»What are the components that make bulk composition
up Coors?
C ? average composition
Quantitative Analysis - how much
is there? spatially resolved
»six pack or case? temporally resolved
»percent ethanol?
Main Topics for Chem 241 Let’s get started

separations Chapter 0 – nice general overview
chemical equilibria - acid/base Chapter 1 – review from 101/102
Chapter 2 – not necessary for this
spectroscopy - the use of radiation class,
l but
b helpful
h l f l for
f 241L
to probe chemical properties
electrochemistry - measurement of
electrical properties of solutions
Section 1 4
Significant Figures Significant Figures - Logs

You should know sig figs
Points will be deducted on exams log 1034 = log 1.034 x 103 = 3.0145
Rounding off is done only on the
FINAL answer log 1.034 x 10-3 = -2.9855
antilog -4.756 = 1.75x 10-5
Accuracy and Precision Accuracy and Precision

Why do measurements disagree? Accuracy - how close to “true” value
Precision - how reproducible
Accuracy and precision not correlated
ERRORS i a measurement can be
i.e. b precise
i andd
not accurate or accurate and not
precise
Types of Errors Uncertainty

Systematic (determinate) - something absolute (ex)
involved with the measurement » estimated uncertainty associated with
system that can be detected and measurement e.g. ±0.2mm
corrected » note that uncertainty has same units as
Random (indeterminate) - natural measurement
limitation in ability to make
measurements
Section 1 5
Uncertainty Propagation of Uncertainty

relative (ex/x)
percent relative (%ex) √
» %ex = (ex/x) (100) √

» for constant absolute uncertainty, the √
relative uncertainty decreases with
increasing magnitude of measurement √
Gaussian Distribution Gaussian Distribution

normal error, or bell shaped, curve
A curve which predicts the
distribution of data - only random
error
MUST define limits of data set

curve characterized by two Arithmetic mean
parameters
» arithmetic mean x
s  xi
» standard
t d d deviation
d i ti x
n
xi  value of measurement i
n  number of measurements
Section 1 6

Standard deviation standard deviation is a measure of the
width of the distribution - how close
   x  x 2 
1
2 the values are clustered about the
mean
s   
i
 n  1 
n  1  degrees of freedom
s 2  variance

standard deviation of the mean Gaussian equation
s
s  1    x  2 2
n y  e 2
 2   
1
2
reduce standard deviation of the
mean by making more
measurements

for x = -  to + , probability must 95.5% of all values fall within ± 2s of
equal 1 the mean
probability that a value falls in some 99.7% of all values fall within ± 3s of
range is the area under curve in that the mean
range Table 4-1, page 56
z = (x-x)/s
Section 1 7
% > x1 x z1 
x1  x
s
y From Table,
Table Find area
for z1 (=Az1)
%= (.5-Az1) x 100
x x1
Gaussian Distribution Confidence Intervals

x x1  x x2  x
Tells us what fraction of time the
% between z1  z2  “true” value will be within the range
x1 and x2 s s
From Table, find area
of our error bars
y f z1 (=A
for ( Az1) and
d z2
(=Az2)
%= (Az1+Az2) x 100
x2 x x1
Confidence Intervals Confidence Intervals

What is the likelihood that the “t” is called “Student’s t” and is a
“true” value (µ) lies in some range statistically derived value
about the mean (x )? the value of “t” is dependent upon the
number of measurements and the
level of confidence desired
ts
x
n
Section 1 8

Increasing the level of confidence
increases the range of values
Increasing the number of
measurements decreases the range of
values
p. 58

Data set (12.6, 11.9, 13.0, 12.7, 12.5) t50% = 0.741
mean = 12.5, std. dev. = 0.4
There is a 50% likelihood that the  0.741 0.4 
true value
l isi in
i what
h range??   12 .5 
5
 12.4 <  < 12.6
 at 90% confidence (t90% = 2.132)
12.1 <  < 12.9
Comparison of Means Comparison of Means

Are two sets of data the same or Comparison of measurement to
different? known value - same as confidence
Calculate the % confidence that they interval
are different - or is difference only
due to random chance x
n  t calc
s
If tcalc > t in Table, data sets are
DIFFERENT at that confidence level
Section 1 9

Comparison of two sets of 1
measurements  x1  x2  n1n2  2
t   
calculate “t”, compare to Table to s  n  n 
determine level of confidence  pooled  1 2 
 
1
  i 1 set 2 j 2
  x x 2   x x 2
 2

spooled   set1 
 n1  n2  2 
 

Comparison of individual differences d
t  
 12
 n 
calculate “t” compare to Table to  sd 
determine level of confidence d  average
g deviation 
 x Ai  x Bi 
n
  d i  d 
1
2
 2
sd   
 n 1 
 
Comparison of Standard Deviations Q test for bad data

s
2 Sometimes, for unknown reasons,
F  12 you get a bad data point
s2 Determine whether it is statistically
s1  s2 significant with Q test
Fcalculated > Ftable means significant difference
Section 1 10
Q test for bad data Q test for bad data

Need at least 4 measurements Gap
The magnitude of Q to reject data is Q
Range
dependent upon # of measurements
and degree of likelihood (Table 4-5)
4 5) Gap = difference between suspect
data point and next closest data point
Range = difference between suspect
data point and furthest data point
Quantification Quantification
Desire linear response with amount can use signal height if peak is
of sample symmetrical - better to use area
plot a parameter of signal (e.g. area, IF detector responds EQUALLY to
height) vs
vs. sample amount (weight
(weight, all analytes,
analytes relative areas = relative
concentration, etc.) amounts
Quantification Calibration Curve

Three common approaches to  Generate a plot of response versus
quantification known amount (e.g. concentration) of
» Calibration (Standard, Working) the analyte of interest
Curve
» Standard Addition x
x
» Internal Standard Response O x
x
x
Amount
Section 1 11
Calibration Curve Standard Addition

Need to minimize matrix differences Add known amounts of analyte to
to reduce matrix effects aliquots of unknown amount
Need stable instrumentation
[ x] A
 x
[ x ]  [ s] Ax  s
 Know [s], measure Ax and Ax+s
 solve for [x]
Standard Addition Standard Addition

Constant matrix - no matrix effects
x Instrument stability still important
x
x
x
[-x]
0 [s]
Internal Standard Internal Standard

Add a known amount of an analyte
(standard) similar to the analyte of [ x] response of x
interest to the sample F
[ s] response of s
Measure response of analyte and
standard F is response factor
Need to know response factor ideally =1
Section 1 12
Internal Standard Internal Standard

Corrects for
x » sample losses during work-up
Rx
x » matrix effects
Rs
x » instrument instability
x
[ x]
[ s]
Calibration Calibration
Sensitivity (m) = slope of Calibration Limit of Detection (LOD) - minimum
Curve detectable amount
 signal 3s
m LOD 
 amount m
Limit of Quantitation
10s
LOD 
m
Calibration Least Squares

many measurements use calibration
curve of response vs. quantity ([ ],
weight, etc.)
get calibration curve collecting data
at several different quantities
Section 1 13
Least Squares Least Squares

assume response is linear and find value of m and b that minimize
uncertainty in response (ey) is >> deviations of y
than uncertainty in quantity (ex) deviation of yi for xi
(standard) di = yi - y
y = mx + b di = yi - (mxi + b)

must square to make all deviations,
positive or negative, weighted the
di 

same
 di2 = (yi - mxi - b)2

y = mx + b

[x]
x   x y   D
Use matrix algebra to determine m
b 
2
and b i i i
x  y
 x y   x
i i
m
i i i
D
D  
x   x 2
y
i i
ni
 xi n
Section 1 14
Least Sq. - Uncertainty Least Sq. - Uncertainty

Because there is an uncertainty in y,
there must be an uncertainty 1
associated with m and b  s 2n  2
sm   y 
 D 
 
  d i  d 2 
1 1
  d i 2
1
  s  x i 2 
2 2 2 2
sy      sb   y

 n2   n2   D 
     
Least Sq. - Uncertainty Least Sq. - Uncertainty

the first decimal place of the standard Uncertainty in quantity of unknown
deviation is last sig. fig for slope or is:
 
intercept
to express uncertainty in terms of y  s y  b   sb 
confidence interval, multiply sm or sb x
by appropriate “t”
m   sm 
Analytical Separations Methods of Separation

 Why separate compounds?  Extraction
» to isolate or concentrate component(s) » washing clothes
from a mixture  Crystallization
» to separate a component(s) from other » drugs
species that would interfere in the
analysis  Distillation
» moonshine
 Chromatography
Section 1 15
Solvent Extraction Solvent Extraction

 Extraction: transfer of a solute from one  Organic solvents less dense than water
phase to another. » diethyl ether, toluene, hexane
 Can use most any combination of phases  Organic solvents more dense than water
( lid liquid,
(solid, li id gas, supercritical
iti l fluid)
fl id) » chloroform, CCl4, dichloromethane
 Solvent extractions use two immiscible  Like dissolves like so ideally, the
liquids. extracting solvent should be similar to
» Typically aqueous/organic solvent combos the solute (analyte)
Separatory
 Solute partitions between the two
funnel phases
add second
immiscible Phase 2 [S]2
solvent
shake Phase 1 [S]1

Equilibrium constant for this  Determination of solute
partitioning is K (partition concentration in each phase
coefficient)
 Define some variables:
[S]2 » V1 & V2 are volumes of solvents 1&2
K= » m = total # of moles of solute (S)
[S]1
present
» q = fraction of solute remaining in
phase 1 at equilibrium
Section 1 16

[S]1 = qm/V1
[S]2 = (1-q)m/V2 K=
[S]2 (1-q)m/V2 (1-q) /V2
= =
[S]1 qm/V1 q/V1
Rearrange:
V1 KV2
q= (1-q) =
KV2 + V1 KV2 + V1
fraction of S in: phase 1 phase 2

 If remove V2 and extract V1 with  Fraction in V1 after n extractions:
fresh layer of V2, what fraction
remains in V1?
» Initial moles = m n
V1
» after first extraction = qm q(n) =
KV2 + V1
» after second extraction = q(qm)=q2m
V1 2
q(2) =
KV2 + V1

 Example: Solute A has a partition a) one 600.0 ml aliquot of hexane is
coefficient of 4.000 between hexane used?
and water. ((K = [S]
[ ]hexane/[S]
[ ]water = 4)) 150ml
If 150.0 ml of 0.03000 M aqueous A q= = 0.05882 = 5.882%
4(600ml) + 150ml
is extracted with hexane, what
fraction of A remains if: # moles remaining
0.05882 (0.03M•0.150L) = 2.647x10-4 moles
Section 1 17

b) 6 successive 100.0 ml aliquots of  Although same volume of hexane is
hexane are used? used, it is more efficient to do several
small extractions than one big one!
6
» 1 - 600 ml extraction extracts 94.12%
94 12%
150ml » 6 - 100 ml extractions extract 99.96%
q= = 0.0004115
4(100ml) + 150ml
# moles remaining
4.115 x 10-4 (0.03M•0.150L) = 1.852x10-6moles
Solvent Extraction (pH effects) Solvent Extraction (pH effects)

 with organic acids/bases:  Partitioning of organic acids between
two phases:
Ka
HA H+ + A- very little here, ions
have poor solubility
Kb HA H+ + A-
organic
B + H2O BH+ + OH-
Generally, neutral species are more soluble Ka
aqueous HA H+ + A-
in an organic solvent and charged species
are more soluble in aqueous solution
 When the solute (acid/base) can total conc. in phase 2

D=
exist in different forms, D total conc. in phase 1
(di ib i coefficient)
(distribution ffi i ) is
i usedd HA [HA]org
instead of K (partition coefficient) D=
K [HA]aq + [A-]aq
Ka
HA H+ + A-
Section 1 18
 Substitute for [A-] in D eq. and [HA]2 [HA]2

rearrange D= 
Ka [ HA]1  K 
[HA]1  [HA]1  1  a 
[H+][A-] [H  ]  [ H ]
Ka [HA]
Ka = [A-] =
[HA] [H+]
[HA]2  K  [HA]2
D= D 1 a  = =K
K [ HA]  [ H ] [HA]1
[HA]1  a  1
[H ]

 pH effect on D for organic acids
 K 
D 1 a  = K
 [ H ] K[H  ] [H+]>>Ka
[H+]=Ka
D pH=pK
pH pKa
[H  ]  Ka K

K K[ H ] mainly mainly
D=  D
 K  [ H  ]  Ka HA A-
 1 a 
 [ H ] [H+]<<Ka
pH

 Example problem: Want to separate  Analogous treatment for organic
two organic acids using a scheme bases (proton acceptors, not KOH)
based on pH. Acid 1 (pKa = 4), Acid
2 (pKa = 8) [ +]]=Ka
[H
pH=pKa [H+]<<K
] Ka
K1 Acid 2 stays in K
K2 organic phase, K Ka mainly mainly
acid 1 is extracted D= D BH+ B
D into aqueous phase [H+] + Ka
[H+]>>Ka
pH
4 pH 8
Section 1 19
Solvent Extraction (pH effects) Separate organic acid, base and neutral
 In general:
Initial Aq. phase
acid base
pH=1, extract with ether
K
Aq. Phase Ether Phase
Org. base Org. acid, Org. neutral
D
extract with pH=12 Aq. Sol’n
Aq. Phase Ether Phase

pH Org. acid Org. neutral
What is Chromatography What is Chromatography

 Method to separate components in a  Chromatography categorized on the
mixture based on different Distribution basis of interaction between solute
coefficients between the two phases. and stationary phase.
 Same principle as solvent extraction,  Mobile phase either gas or liquid
but one phase is stationary and one  Stationary phase either liquid or
phase is “mobile” solid
 Stationary phases are most commonly
coated or packed in a column
What is Chromatography What is Chromatography

Liq/Liq (Partition) Liquid  Other modes of chromatography
Liq/Solid (Adsorption) Chromatography (Fig. 23-6)
Gas/Liq (Partition) Gas » Ion-exchange: separates charged
species
Gas/Solid (Adsorption) Chromatography
» Size exclusion or Gel Permeation:
separates molecular size
» Affinity: separates on the basis of
antibody-anitgen, enzyme-substrate
interactions
Section 1 20
Chromatography Basics Chromatography Basics

 Typical chromatogram: tr2
tr1
ponse
Dettector
tm
ttr1
Resp
Response
Detector
injection time time or volume

tm = time for mobile phase to travel tr = retention time
time or volume length of column (dead time)
tr = adjusted retention time
= tr - tm  = tr2/tr1 = relative retention

Mobile phase flow rate:  Partition coefficient K = Cs/Cm
» volumetric flow rate (F): ml/min » C = Concentration of analyte
» linear flow rate (v): cm/min (mm/min) » s = stationary phase
Two
T ways to describe
d ib solute
l » m = mobile
bil phase
h
“retention”  Vs = volume of stationary phase
» retention time, tr Vm = volume of mobile phase
» retention volume, Vr
Vr = Ftr

 Capacity factor, k: a measure of What fraction of time does the solute
retention spend in mobile phase?
» higher k = greater solute retention  q = fraction of solute in mobile
phase
Vs moless
k = K =
Vm molesm
ts tr - tm
k = t = t
m m
Section 1 21

molesm
q = moles + moles
C V
= C V m +mC V  Fraction of time solute spends in
s m m m s s mobile phase
1
q = 1 + kk
1 1
q= CV q= Vs  larger k means greater retention times
1+ s s 1+K
CmVm Vm  Fraction of time solute spends in
stationary phase = (1-q)
Vs
K = capacity factor = k k
Vm (1-q) = 1 + k

 Rate of travel of solute molecule  Retention time, tr: time it takes
through column: solute to go from beginning to end
linear flow rate (cm/min) of column. column length
L
rate = v (fraction of time in mp) tr = rate of solute travel
1 1 L
rate = v =v
tr = tm = L
Vs 1 v
1+K 1 + k v
Vm Vs
1+K
Vm
Chromatography Basics Efficiency of Separation

 Retention time, tr:  Typical chromatogram:
Vs
tr = tm ( 1 + K ) = tm (1+k’)
Vm
 Retention volume, Vr: multiply
Response
Detector
retention time by volumetric flow rate, F

Vs
Vr = Vm ( 1 + K ) = Vm + KVs time or volume
Vm
Section 1 22
Efficiency of Separation Efficiency of Separation

 Two factors affect how well two  Gaussian peak shape:
components are separated
» difference in retention time
» peakk widths
idth
 Solutes in a column spread into a  
w1/2=2.35
h
Gaussian profile: 1/2h
w=4
t0 tr
time

 The resolution (separation) of two  Resolution: higher R, better
solutes: separation
tr Vr R=0.50 R=0.75
» resolution = w =w
avg avg
t0  time t0  time
tr = difference between retention times of two peaks
R 1 R=1.00 R=1.50
tr = (tr2 - tr1)
is good
wavg = average of the peak widths at baseline ( 4)
t0  time t0  time

 Plate theory: tr
16 t r
2
5 . 55 t r
2 2
» treats separation in discrete stages, N   

more stages = more plates.  2
w 2
w1 2
2
Theoretical
Th ti l plates
l t (N):
(N) a number
b N is
i specific
ifi ffor eachh solute
l on a
indicating how good a column is for given column
a separation Increasing retention time increases N
Section 1 23

 relation of N to Resolution (R):  N required to obtain a certain
resolution: 2
16 R
N
R
N
  1   12
4
N1 N2 N2>N1
tr2
=
tr1
t0 time t0 time
Efficiency of Separation Why Bands Spread

 N depends upon the length of the column  Band broadening
 Independent of the column length is the
Height Equivalent of a Theoretical Plate
 Causes of band broadening:
» Longitudinal diffusion
L Lw2
HETP   » Resistance to mass transfer (RMT)
N 16tr 2 » Eddy diffusion
As HETP , resolution increases (N )
Why Bands Spread Why Bands Spread

Longitudinal diffusion: solute [ ] is Resistance to mass transfer (RMT):
lower at the edges of a band; solute Mobile
diffuses to the edges. phase slow equil.
Stationary
St ti
phase
bandwidth bandwidth
Low High Low
Same Conc.at Equil.
Conc. Conc. Conc.
HC = Cv : C = constant, v = flow rate
HB = B/v : B = constant, v = flow rate decrease HC by decreasing v
decrease HB by increasing v
Section 1 24

Eddy diffusion (not simple diffusion):
Van Deemter Equation:
HETP = HA + HB + Hc
HETP = A + (B/v) + Cv
time
HA = A : A = constant, depends on size of

particles

 van Deemter Plot: Asymmetric peak shapes: K depends
on [ ] at high [ ] (solute becomes
solvent)
H B Cv
(+) overloaded
Linear ideal
v peak shape
Hmin
Cs
(-) tailed
A
vopt
Cm
v
Why Bands Spread General Chromatography

 Asymmetric peak shapes Chromatography
Observed
slow slow chromatogram Gas Liquid
(+) deviation:
fast
GSC GLC LSC LLC IEC GPC AC
fast fast
fast fast
GC: volatile solutes
(-) deviation:
slow slow LC: any mobile phase soluble solutes
slow
time
Section 1 25
Gas Chromatography Gas Chromatography

 Gas chromatography:
» Analytes (volatile) are vaporized and
transported through the column
» Gaseous mobile phase: (He(He, N2, or
H2)
 as long as mobile phase is inert, choice
is not critical
» Stationary phase: non-volatile liquid
or solid particles
Gas Chromatography Gas Chromatograph

 Schematic diagram of GC:
carrier gas detector
septum
exit
injection
port
column
column oven
Tinj,det  Toven + 50°C
Gas Chromatograph Gas Chromatograph
Section 1 26
Gas Chromatograph Gas Chromatography

 Solid stationary phase:
» fine solid particles packed into
stainless steel tubing
 carbon
b (carbon
( b bl black)
k)
 SiO2 (silica gel), Al2O3 (alumina)
» analyte adsorbs directly on solid
particles
» gives strong retention of solutes
(large surface area)

 Liquid stationary phase:  Liquid stationary phase cont’d:
» non-volatile liquid coated on inside of » ideal characteristics - wide liquid range
column or on fine solid support (min/max temp limits), stable, low
volatility, low viscosity (D and RMT)
» ideal characteristics of solid support: » examples of liquid stationary phases :
 strong, porous, high surface area and silicones: non to strong polarity depending
inert (non-adsorptive) on R groups
» real example of solid support: R R
 diatomaceous earth (algae skeletons) O Si O Si
R R n

 Liquid stationary phase cont’d:  Columns: packed
» ~ 3-6mm inner diameter
» examples of liquid stationary phases tubing, 1-5 m long
 carbowax: strong polarity » used for preparative
—CHCH2CH2O)n— separations
i or to separate
 other examples in Table 24-1 gases that are poorly
retained
» chose stationary phase to match » lower resolution
polarity of solute (like dissolves like) » small, uniform particle
 solute polarities listed in Table 24-2 size decreases Eddy
diffusion (requiring
higher pressures)
Section 1 27

 Columns: open
tubular
» 0.1-0.5 mm inner dia.,
10-100 m long
» 0.1-5 m thick sp
coated on inner walls
» higher resolution,
shorter analysis times,
greater sensitivity
compared to packed
columns
Detectors Detectors
 Thermal Conductivity (most
common): e-
gas
He has high thermal conductivity, is main

component entering detector
Detectors Detectors
 Thermal Conductivity
 Thermal Conductivity
e-
» universal
» non destructive
non-destructive
gas
» linear range (>105)
» Detection limit is  400 pg
analyte appears, T.C., Temp , Wire
Resistance , detector current   “read-
out”
Section 1 28
Detectors Detectors
 Flame Ionization Detector  Flame Ionization Detector (FID):
(FID): » organic solutes are burned in flame
cathode (collects producing CH radicals and eventually
CHO+ ions) CHO+
anode . .
» CH + O  CHO+ + e-
air » CHO+ ions are collected by cathode,
H2 produces current as the response
column effluent
Detectors Detectors
 Flame Ionization Detector (FID):  Electron Capture (ECD):
» organics (reduced carbon only) Radioactive
» Destructive -emitter insulator
7) Ni63 - +
» li
linear range (>10
(
» detection limit is  2 pg
- +
electrodes
Detectors Detectors
 Electron Capture (ECD):  Electron Capture (ECD):
» -
+ gas  + + - gas+
(standing e- » Non-destructive
current) » non-linear
» e- + solute  solute- (e- capture) » l i to e- capturing
selective i solutes
l
» solute- + gas+  solute + gas » detection limit  5 fg
Current (I)
standing
current
time
Section 1 29
Detectors Qualitative Analysis

 Variations of FID:  All previous detectors non-specific
» Flame Photometric  retention times alone can’t identify a
» Alkali Flame compound
» Sulfur
S lf Chemiluminescence
Ch il i » Mass spectrometer
 Characteristics in Table 24-5 » Fourier Transform Infrared Spectrometer
» compare spectra obtained by these two
detectors with known sample spectra
(fingerprint)
Qualitative Analysis Qualitative Analysis

Problems with comparing retention Kovats retention index (I):
times for qualitative analysis:
» tr dependent upon:   log trx  log trn 
I  100 n  ((N - n)) 
l trn 
L
v  l trN  log
 log
K f(temp) n = # of C atoms in smaller alkane
Vs, Vm f(packing) N = # of C atoms in larger alkane
» Can compare tr to retention of trx = adjusted retention time of unknown
standard solutes, typically alkanes trn = adjusted retention time of smaller alkane
trN = adjusted retention time of larger alkane
Qualitative Analysis Qualitative Analysis

Kovats index for linear alkanes Using Kovats retention index:
equals 100 times the number of
carbon atoms
  log trx  log trn 
I  100 n  (N - n) 
C8
C9   log t   log t 
rn  
Response
C7 rN
Detector
air x
  log (6.3 - 0.25)  log(5.6 - 0.25) 
I  100 8  (9 - 8) 
  log (7.4 - 0.25)  log(5.6 - 0.25) 
0.25 3.5 5.6 6.3 7.4
Time (min) I = 842
Section 1 30
Temperature Programing Liquid Chromatography

 Preparative LC- separates
milligrams to grams of analyte
Response Response Response
C6 C7 Low T
air C8 C9
Analytical - separate micrograms to
picograms - HPLC
R
C6 C7 C8 C9 C10 High T High Performance LC

air C11 C12
» Liquid/Solid (LSC)
C6 C7 C8 C9 C10 C11C12 C13
» Liquid/Liquid (LLC)
T
air
time
Liquid Chromatography Liquid Chromatography

 Small diameter packing (stationary
phase):
» provides more uniform flow (A ) 60 10m
» less distance for solutes to diffuse in 40 Packing
H (m))
mobile phase to interact with stationary 20 5m diameter

phase (C ) 10
3m
» sacrifice: much higher pressure is
required to “push” mobile phase through 0 2 4 6 8
column (~3000psi = 200 atmospheres) Flow rate (ml/min)

 Liquid-Solid chromatography
(LSC): (adsorption)
» Stationary phases: very porous =
high surface area for interaction with
mobile phase
 silica gel: SiO2 xH2O
alumina: Al2O3  xH2O
Section 1 31

Silica - OH groups very polar  Liquid-Solid chromatography (LSC):
» Mobile phases: solvent displaces solute
OH OH from stationary phase, rather than solute
partitioning
O Si O Si 5SiO2  2H2O » Elutropic series: relative ability of solvent
O O n to displace solute (Table 25-2)
O Si O Si  Methanol>acetonitrile>chloroform>hexane
O OH » The greater the eluent strength, , the
O Si OH more rapidly solutes will be eluted

 Elutropic elution:
Toluene °=0.22
“isocratic
elution”
Acetonitrile °=0.52
Start w/ 100% Benzene “gradient

then add Acetonitrile
elution”

Gradient elution - composition of the  LSC:
mobile phase changes with time » can use thin-layer plates (TLC):
solvent A = benzene, solvent B =
methanol
%A
cheap, easy to do and learn, simultaneous

separations, but poor quantification,
time reproducibility and resolution
Section 1 32

 Liquid-liquid chromatography  Common liquid stationary phases:
(LLC): » Polar phases:
» Stationary phase: viscous liquid  Amino [R = (CH2)3NH2 ]
coated or chemically bonded to  Cyano [R = (CH2)3CN ]
exposed silanol groups of silica (solid  Diol [R = (CH2)2OCH2CH(OH)CH2OH ]
support) CH CH
» Non-Polar phases:
3 3
 Octadecyl, C18 [R = (CH2)17CH3 ]
Si OH + Cl Si R Si O Si R  Octyl [R = (CH2)7CH3 ]
CH3 CH3  Phenyl [R = (CH2)3C6H5 ]

 “Normal” phase LC: “Reversed” phase LC:
» Polar stationary phase » Non-polar stationary phase
» Non-polar mobile phase
» non polar  polar
Order of elution: non-polar
» Polar mobile phase
p
» Solvent strength: non-polar (weak), polar » Order of elution: polar  non-polar
(strong) » Solvent strength: non-polar (strong),
polar (weak)
Like dissolves Like! Like dissolves Like!
Liquid Chromatography LC Instrumentation

 “Classical” vs HPLC:  Schematic diagram of HPLC:
Packing Pressure N
Pump Injector
classical: 50 m gravity 500
HPLC 3-5
HPLC: 3 5 m 3000psi
3000 i 15,000
15 000 Pump I
Column Detector
» HPLC: higher resolution, faster and Solvent Solvent To waste or
good quantification, but expensive and A B fraction collector
complex isocratic: 1 solvent
gradient: 2 or more solvents
Section 1 33
LC Instrumentation LC Instrumentation
Injection valve  Primarily use packed columns
from pump although capillary columns are
to column gaining in popularity
» 5-30 cm long
» 1-5 mm i.d.
waste
Injection port » Often use a guard column to protect
Sample loop main column
LC Instrumentation LC Instrumentation
 UV spectrophotometric Detectors:  UV Spectrophotometric Detectors:
» linear (105 range in solute [ ] )
Eluate in » 0.1-1 ng detection limit
» f i l universal
fairly i l (solvent
( l limited)
li i d)
Light » can handle gradients
Photo
source Detector  Fluorescence Detectors are 100x
more sensitive, but less universal
Eluate out
LC Instrumentation Commercial LC Instrument

 Mass Spectrometer Detectors:
» linear (105 range in solute [ ])
» sensitive (0.1-1 fg detection limit)
» can handle
h dl gradients
di
» universal
» response is fairly specific for each
compound
» often used in conjunction with UV det.
Section 1 34
Ion Exchange Ion Exchange

Partition solute between solvent and Stationary phase is a cross-linked
charged sites on stationary phase copolymer
» solute must be ionic » vinyl benzene/divinylbenzene
O
Opposites
it attract
tt t » vinyl
i l benzene
b  polystyrene
l t
» anion exchange - positively charged » divinylbenzene is cross linker - links
stationary phase polystyrene chains together
» cation exchange - negatively charged
stationary phase

Stationary phase Stationary phase - cross linked
polymer CH CH CH CH CH
CH CH2 CH CH2 2 2
CH CH2 CH CH2 CH CH2 CH

CH CH2
Vinyl benzene
divinyl benzene
Use 1 - 16% divinyl benzene, amount changes pore
size, rigidity - cross linking,  selectivity R
Section 1 35

Binding of analyte depends upon Selectivity coefficient
» Magnitude of charge
Coulombs Law - F  q1q2/r2 R-Na+ + Li+ R-Li+ + Na+
3+ >2+ > 1+
» size of hydrated ion (related to r in [ R  Li  ][ Na  ]
Coulomb eq.) K
[ R  Na  ][ Li  ]
Selectivity coefficients relative to Li = 1.00

Gradient elution with increasing ionic
strength or pH change
KAg > KNa
» IIncreasing
i solvent
l [Li+] elutes
l N +
Na
before Ag +
» Note that Li+ and H+ are the most

weakly bound ions, but can still use
them to elute other ions if their [ ] is
sufficiently high
Ion Exchange Molecular Exclusion

More commonly called Gel
Permeation Chromatography (GPC)
Also called Size Exclusion
Chromatography (SEC)
Section 1 36
Molecular Exclusion Molecular Exclusion

Stationary phase contains small pores
that analytes can diffuse into
(depending upon size)
Larger molecules cannot fit into
pores so they elute faster than smaller
molecules

 Stationary Phase  Stationary phase typically a cross-linked
» pore size determines range of MW polymer
which can be separated (see Table 26-  Common stationary phases
4) » Sephadex (glucose/glycerol polymer,
polymer Fig.
Fig
» exclusion limit: smallest molecule 26-2)
which can not fit into the pores; any » Bio-Gel (polyacrylamide gel, Fig. 26-12)
larger molecule will have same Vr

Stationary Phase - solvent trapped in
pores (Vs) Vr  Vm  KVs
Mobile Phase - solvent outside of Vr  V0  KVs
pores
Mobile Phase Volume (Vm) = Void V  V0
K r
Volume (V0) Vs
If solute is larger than pores,
Vr = V0 and K=0
Section 1 37

For intermediate sized molecules,
VT  V0  Vs
0 < K < 1
Vs  VT  V0 If K > 1, analyte has adsorped to sites
Vr  V0 on column
l
K av  VT = volume of column
VT  V0
If solute is small and penetrates all pores,
Vr = VT and K=1

Applications of GPC  Molecular weight determination:
» Molecular weight determination: » compare Vr of unknown to that of
 separation based on hydrodynamic standards with known MW and
radii which
hich is roughly
ro ghl correlated to MW similar str
structure
ct re
» Desalting - remove salt impurities
from biological samples 6 Different classes
Log MW of stationary
4
phase
2
Vr
Affinity Chromatography Affinity Chromatography

 Makes use of tight, specific complex  Process
(highly selective):
» antigen/antibody
» enzyme/substrate
/ bt t support
linker
 Very powerful method of arm affinity
ligand
purification in biology
Section 1 38
Affinity Chromatography Electrophoresis

 Can release bound species by  Separation is based on differences in
changing pH, temp, ionic strength, migration of charged ions in an
etc. electric field in solution
 Example chromatogram: typically carried out on buffer soaked
“garbage”
paper or gel
change
conditions desired solute
+
(+) anode n (-) cathode
- ++
Electrophoresis Electrophoresis
 Rate of migration dependent upon: Rate of migration:
 size of solute » electrophoretic velocity, Vep = epE
» larger size, more friction, slower ep: electrophoretic mobility = q/f
movementt q = charge
h iin Coulombs
C l b
 charge of solute f = friction coefficient (f = 6r, Stokes
» greater charge, increased force Eq.)
(Coulomb’s law), faster migration E = electric field strength (volts/cm)
Capillary Electrophoresis Commercial CE instrument

 Schematic:
flow
fused silica
capillary
50cm
HV pressurized
(30kV)
Section 1 39
Capillary Electrophoresis Capillary Electrophoresis

 Fused silica capillary:  electroosmotic flow:
» fused silica has exposed silanol
polyimide groups (Si-OH); deprotonated at pHs
coatingg
15m thick
fused silica 2
330m dia.
» Causes a layer of + charge to build up
at surface
+- +- -+ -+ - +- +- +- +- + - + - + -+ +
-
O- O- O- O- O- O- O- O- O- O- O- O- O- O- O- O-
opening (+) flow (-)

anode + +- O-+ cathode
25-75m dia. O-- O- - +O-- O+-- O+
- O- - -O- +O-- O+-- O+- -O-+O-- + + -+-
O-- O- O

 electroosmotic flow:  electroosmotic flow velocity
» surface charges move to cathode,  Veo = eoE:
dragging solution
» eo: electroosmotic mobilityy (( surf.
» surface charge “removes”
removes friction charge dens.  1/(ionic strength)1/2
between solvent and walls of capillary
» E = electric field strength (volts/cm)
laminar  apparent mobility: app = ep + eo
electroosmotic flow profile hydrodynamic flow profile  apparent velocity: Vapp = appE
Vapp Ld t Ld Lt Veo Vep Vapp

 app   
E V Lt Vt
Ld = length of column from injector to detector +

t = migration time (+) anode n (-) cathode
Lt = total length of column -
V = voltage applied to column
Section 1 40

 Efficiency:
L2
» no particles so no multiple paths (A = 0) N   2 Dt 1 2
» no stationary phase so no RMT (C = 0)  2
» HETP = B/v Vapp Ld t Ld Lt

» can increase velocity by  applied voltage,  app   
but due to resistance, this generates heat E V Lt Vt
which  longitudinal diffusion (B)
appV
» still get high efficiencies (N=105-106) N
2D

Sample injection
» Very small sample sizes - 10-9 liters
» Hydrodynamic injection, uses pressure
to force sample onto column
» electrokinetic - based on ep thus
injected sample has different relative
composition of analytes
Capillary Electrophoresis
 Detection:
» UV/Fluorescence
» Mass Spectrometry
Characteristics
Ch i i off CE
» very fast
» high efficiencies
» especially useful for biopolymers
Section 1 41

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Chem 241 - Summer 2010

Chemistry 241 About Me

Chemistry 241 Prerequisites Class logistics

Class logistics Class logistics – Schedule

May 17 Chpt. 25,26 Chpt. 9 Chpt. 10 No Class

May 24 No Class No Class No Class No Class No Class

Class logistics - Exams Honor System (http://honor.unc.edu)

Honor System Procedures Exam Policies

Exam Policies Class logistics - Grading scale

Class logistics - Alternative scale Class logistics - Equations

[ H+ ] + [ H+ ] Ka1 + Ka1 Ka2

[Base] - b  (b2 - 4ac )

The scale that gives you the highest grade Csolute

will be used Cstandard areastandard

Class logistics - Homework Course Goals

What is Analytical Chemistry? What is an “Analyte”

Analytical chemistry is object of the chemical

What are we trying to measure? Define what is being measured

Main Topics for Chem 241 Let’s get started

Significant Figures Significant Figures - Logs

antilog -4.756 = 1.75x 10-5

Accuracy and Precision Accuracy and Precision

Types of Errors Uncertainty

Uncertainty Propagation of Uncertainty

» %ex = (ex/x) (100) √

Gaussian Distribution Gaussian Distribution

Gaussian Distribution Gaussian Distribution

Gaussian Distribution Gaussian Distribution

Gaussian Distribution Gaussian Distribution

Gaussian Distribution Gaussian Distribution

Gaussian Distribution Gaussian Distribution

Gaussian Distribution Confidence Intervals

Confidence Intervals Confidence Intervals

Confidence Intervals Confidence Intervals

Confidence Intervals Confidence Intervals

Comparison of Means Comparison of Means

Comparison of Means Comparison of Means

Comparison of Means Comparison of Means

Comparison of Standard Deviations Q test for bad data

Fcalculated > Ftable means significant difference

Q test for bad data Q test for bad data

Quantification Calibration Curve

Calibration Curve Standard Addition

Standard Addition Standard Addition

Internal Standard Internal Standard

Internal Standard Internal Standard

Calibration Least Squares

Least Squares Least Squares

Least Squares Least Squares

Least Squares Least Squares

Least Sq. - Uncertainty Least Sq. - Uncertainty

Least Sq. - Uncertainty Least Sq. - Uncertainty

Analytical Separations Methods of Separation

Solvent Extraction Solvent Extraction

Solvent Extraction Solvent Extraction

shake Phase 1 [S]1

Solvent Extraction Solvent Extraction

Solvent Extraction Solvent Extraction

fraction of S in: phase 1 phase 2

Solvent Extraction Solvent Extraction

Solvent Extraction Solvent Extraction

Solvent Extraction Solvent Extraction

Solvent Extraction (pH effects) Solvent Extraction (pH effects)