Tutorial 8

Instrumental Analysis (I)

HPLC
 Graded presentation
Students in groups of 4-5 individuals are asked to prepare a presentation
(weight=5% of the theoretical course assessment) during the session of the last
tutorial of chromatography due from 25 to 28 November. Each group will be
asked to give a 10 min talk about the presentation and has to be prepared for
students and instructors questions and discussions. Each group will have a topic
according to the coming slide. The assessment criteria are:

Criterion Grades (out of

10)
Choice of the topic 2
Content of the presentation 2
Comprehension of the content by members of the 2
2

group
Quality of the presentation 2
Ability to answer students and instructors 2
questions
 TOPICS FOR PRESENTATIONS due after next
tutorial (last tutorial of chromatography)
5% OF THE TOTAL GRADE

Group Topic
1 Protein analysis by HPLC
2 Types of stationary phases in Chiral Chromatography
3 UPLC
4 Monolithic columns and new trends in stationary phases
5 Reversed stationary phases
6 Softwares in chromatography
7 Theories
8 Analysis of water/fat soluble vitamins
9 Gas chromatography applications
10 Supercritical fluid chromatography applications
 Today's Objective

Describe principles of separation in chromatography.

Estimate the tailing factor Tf.
Assess chromatograms visually by comparison.
Calculate chromatographic parameters from given data.
Assess several chromatographic experiments by calculating
parameters from given data
Solve a gradient elution problem.
Solve an upscaling problem.
Next time, the instructor will see your presentations!!!
The details of the presentation requirements
and assessment are in the last slide of lecture 6.
 Tailing factor Tf = a+b / 2a

Tf = 1 Tf > 1 Tf < 1
 Useful Equations in Chromatography
Cs d substance
K : partition coefficient RF : RF value
Cm d solvent front (in planar chromatography)

I t ' Vs ts
k' R K
tM Vm tm
: capacity factor

tR ' tR tM
tR'= tR2'= time
tM tR1 tR2
tR1- tM tR2- tM

Separation factor t ' R 2 t ' k '2 k ' K 2 K

R1 1 1
 Useful Equations in Chromatography

L 2
R
N 1



k '2

HETP H 4 1 k 'av
N x 1 k 'av
2

N 4 R
2 2 1 k '2
16 t R 5.54 t R
N 2
2
w w0.5 t R VR
R
wav wav
Linear flow rate (ux)= L / tM
 Compare Chromatograms
#1
Chromatograms of compounds A and B were obtained at the same flow
rate with two columns of equal length.

1. Which column has more theoretical plates? 1

2. Which column has a larger plate height? 2
3. Which column gives higher resolution? 1
4. Which column gives a greater relative retention? neither
5. Which compound has a higher capacity factor? B
6. Which compound has a greater partition coefficient? B
 Simple Numerical Problems -1
#2
A chromatography column with L = 10.3 cm and ID = 4.61 mm is packed
with a stationary phase that occupies 61.0% of the volume. The linear
flow rate = 17.4 cm min-1.
1. How long does it take for solvent to pass through the column?

tM = L / ux = 10.3 cm / [17.4 cm min-1] = 0.592 min.

2. Find the retention time for a solute with a capacity factor of 10.0.

k' = t'R / tM = (tR-tM) / tM

=> tR = k' tM + tM = tM (1 + k') = 0.592 min x 11 = 6.51 min.
 Assess a Chromatographic System -1
#3
A chromatogram with ideal Gaussian bands has tR = 9.0 min and w0.5 =
2.0 min.

1. How many theoretical plates are present?

N = 5.54 tR2 / w0.52 = 5.54 * 92 min2 / 22 min2 = 112.

2. Find the plate height if the column is 10 cm long.

H = L / N = 10 cm / 112 = 0.89 mm.

 Predict the Peak Width
#4
A band having a width of 4.0 mL and a retention volume of 49 mL was
eluted from a chromatography column.
N.B. Assume that the only band spreading occurs on the column itself.

What width is expected for a band with a retention volume of 127 mL?

from the known:

N = 16 tR2 / wminutes2 = 16 VR2 / wvolume2 = 2401
is the number of plates for this column in this experiment.
for the unknown:
wunk2 = 16 VR-unk2 / N = 16 * 1272 mL2 / 2401
wunk = 10.4 mL. Chart parameters like retention, peak width,
etc. can always be expressed in units of
volume, time or cm resp. mm.
If you apply rules make sure that you have all
of them in suitable dimensions.
 Assess a Chromatographic System -2
#5
Two compounds with partition coefficients of 0.15 and 0.18 are to be
separated on a column with Vm / Vs = 3.0.

Calculate the number of theoretical plates needed to produce a

resolution of 1.5. K
2 0.18 / 0.15 1.2
N 1 k '2 K1
R
4 1 k 'av Vs
k '2 K 2 0.18 / 3 0.060
2
Vm
1 k 'av Vs
N 4 R k '1 K1 0.15 / 3 0.050
Vm
1 k '2

N = [6 * 1.2/0.2 * 1.055/0.06]2 = (400689 =) 4 x105.

 Simple Numerical Problems -2
#6
An open tubular column (OTC) with L = 30.1 m and ID = 0.530 mm is
coated on the inside wall with a layer of stationary phase that is 3.1 m
thick. Unretained solute passes through in 2.16 min, whereas a particular
solute has a retention time of 17.32 min.

Find the capacity factor for the solute and the fraction of time spent in the
stationary phase.
k' = t'R / tM = (tR-tM) / tM = (17.32 min 2.16 min) / 2.16 min = 7.02.
k' = ts / tm => ts = k' * tm = 7.02 x 2.16 min = 15.16 min.
fraction in stat. phase = 15.16/17.32 = 0.875

Find the partition coefficient Cs/Cm for this solute.

K = Cs / Cm
k' = K Vs/Vm
=> K = k' * Vm / Vst = k' * (L*Amob)/(L*Ast) = k' Amob / Ast
Ast = Atotal - Amob = D2/4 - Dmob2/4
= /4 [(530 x10-6 m)2 (523.8 x 10-6 m)2]
= 5.13 x10-9 m2
K = k' * Amob / Ast = 7.02 * 2.15 x10-7 m2/ 5.13 x10-9 m2
= 294.
 A Problem on Gradient Elution
#7
A mixture of 14 compounds was subjected to a RP gradient separation
going from 5% to 100% acetonitrile (CH3CN) with a gradient time of 60
min. All peaks were eluted between 22 and 41 min.
1. Is the mixture more suitable for isocratic or gradient elution?

tG = 60 min, t = 41-22 = 19, t / tG = 19/60 = 0.317

Use gradient elution if t / tG > 0.25

Use
2. If theisocratic
next runelution if t / tselect
is a gradient, G < 0.25
the starting and ending %age
acetonitrile and the gradient time.
equation of the graph %age ACN vs. time:
%ACN = (100-5) % / (60 min) * X min + 5%
%ACN at 22min: %ACN = 95/60 %/min * 22 min + 5% = 40%
%ACN at 41min: %ACN = 95/60 %/min * 41 min + 5% = 70%
Thus, a suitable gradient would be from 40 to 70% ACN over 60 mins.
 A Problem on Peak Shape
#8
Suppose that an HPLC column produces Gaussian peaks. The detector
measures absorbance at 254 nm. A sample containing equal moles of
compounds A and B was injected into the column.
(AUC = 1.064 x h x w0.5)
compound >
V parameters A B

254 2 x 104 M-1 cm-1 1 x 104 M-1cm-1

peak height 100 mm ?

peak half-width 10 mm 12 mm

What is the peak height of B?

AUC = 1.064 x w0.5 x h.

AUCa .
=> AUCA / AUCB = A / B

AUCB = AUCA x B / A.

AUCA = 1.064 * 100 mm * 10 mm = 1064 mm2.

AUCB = AUCA x B / A = 1064 mm2 x 1/2 = 532 mm2.

hB = AUCB / (1.064 x w0.5B) = 532 mm2 / (1.064 x 12 mm) = 41.6 mm.

 "Rules of Thumb" for Up- (or Down-)Scaling
CASE I CASE II
Suppose you want to separate a mixture by Suppose there is an instruction telling you
chromatography without having a suitable how to separate 100 mgs of your mixture by
instruction. So in order to find a suitable a chromatographic method. In fact, you
method you try several systems with small have only 20 mgs of this mixture to be
samples of the mixture. separated.

What do you have to consider if you want

to apply the found method to your mixture?

1. Keep column length constant (if separation is o.k.).

2. Cross-sectional area of column mass of analyte:
2
mass 2 radius 2

mass 1 radius 1
3. Sample volume applied to column mass of analyte.
 A Problem on Upscaling
#9
A chromatographic procedure separates 4.0 mg of unknown mixture on a
column with a length of 40 cm and a diameter of 0.85 cm.

1. What size column would you use to separate 100 mg of the same mixture?
same length (40 cm),
diameter: mB / mA = rB2 / rA2 = dB2 / dA2
dB2 = dA2 * mB/mA = 0.852 cm2 * 100 mg/4 mg = 18.06 cm, dB = 4.25 cm
 Separation Principles
# 10
Match terms in the 1st list with the characteristics in the 2nd list.
A. Ions in mobile phase are attracted to
counterions covalently attached to
1. adsorption chromatography C stationary phase
2. partition chromatography D B. Solute in mobile phase is attracted to
specific groups covalently attached to
3. ion-exchange chromatography A
stationary phase
4. molecular exclusion
E C. Solute equilibrates between mobile
chromatography
phase and surface of stationary
5. affinity chromatography B phase
D. Solute equilibrates between mobile
phase and film of liquid attached to
stationary phase
E. Different-sized solutes penetrate
voids in stationary phase to different
extents.