High Performance Liquid Chromatography: Barba, Biadomang, Dulos CHEM 127.1

High Performance
Liquid Chromatography
Barba, Biadomang, Dulos
CHEM 127.1
Saturday, October 8, 2011
Caffeine
belongs to a group of
alkaloids called xanthines
sample in this
experiment
beverage
HPLC
Before: large columns, large particles, under

gravity feed, manual collection of fractions of
eluents
Giddings Prediction: smaller particles, under

HPLC
Csaba Horvath and

colleague S.R. Lipsky
built rst HPLC
But, in 1970s
small silanized
particles allowed use
of small-volume
columns=high
resolution
Principles
In LC, rate of solute distribution between

S.P and M.P
diffusion-controlled
to minimize diffusion and time required for

the movement of sample and from
interaction sites in the column
smaller eddy diffusion (small A value)

HPLC
B is usually small for

liquids
small molecular
diffusion
A is usually small and

almost constant for
liquids
Stationary phase
HPLC microparticles: high-purity silica
low in trace metal content
5 to 10 micrometers in diameter
pore sizes: 60 to 100 angstroms, 300 for

large biomolecules
It can be partition (liquid-liquid) or

adsorption
microporous
10 micrometers
permeable to solvent
e.g. silica gel
perfusion
12 micrometers
Pump
forces the mobile phase
specic ow rate
isochratic or gradient
Injector
introduces liquid sample

Pump
Isocratic
preparative
Gradient
analysis
Pump
Reversed phase
nonpolar S.P. polar M.P.
S.P: aromatic or heterocycles to provide to

interactions
dispersive forces
Normal phase
polar S.P. nonpolar M.P.

Spectroscopic
UV absorption
MS detection
Refractive Index
RI: measure of molecules ability to

deect light
HPLC
HPLC can be used for quantitative and qualitative

analysis
preparation of pure compounds

HPLC
HPLC demonstration
Methodology
Preparation of Caffeine
Standard
lor dlerenL concenLrauons of
caelne soluuons
3 100mL vol asks. Welgh 2.3,
3.0, 7.3 and 10.0 mg caelne

dlluLe Lo mark wlLh
meLhanol:P
2
C (2:8) (M),
ad[usL pP=3 wlLh P
3
C
4
phosphaLe buer-conLrol pP, neuLrallze
charges on S and neuLrallze charges on
analyLe
solvenL-polar, commonly used wlLh C
18
columns
MeLhanol: n modler
MlxLure: waLer, meLhanol assoclaLed wlLh
waLer
shake, degas (3mlns), lLer
(0.43m lLer membrane)

Shaklng - ensure dlssoluuon
uegasslng - P
2
C has hlgh dlssolved
gas conLenL, come ouL and be
enLrapped, aecL reLenuon ume
lllLer - remove oLher parucles and
unlformlLy of slze
Cn=pump and deLecLor,
pump ow raLe=2.3mL/mln,
deLecLor
sensluvlLy=0.08AulS

Cn=recorder, slow speed raLe,
prlor ln[ecuon, allow M Lo
pass (3-10mlns),
slmulLaneously record deLecLor
response

1o sLarL readlng
AulS = absorbance unlL full scale
w/ syrlnge, ln[ecL 23L
caelne sLd sLarung wlLh
leasL concenLraLed. 1ake
dupllcaLe chromaLograms
for dl sLds

Lnsure no lnLerferlng subsLances are
presenL ln Lhe column
S: Slllca bonded Lo alkyl chaln (C
18
)
- non polar, hydrophoblc
1o obLaln sLandard
chromaLograms
Determination of Caffeine in Coffee and
Measure amounL of caelne
ln coee

0.3mL coee ln 23mL vol
ask

0.3mL Lea ln 23mL vol ask

Measure amounL of caelne
ln Lea

dlluLe Lo mark w/
meLhanol:waLer (2:8)

Same solvenL sysLem
lollow same sLeps ln A

Determination of Caffeine in Cola
Beverage
Cease bubbllng
10mL cold beverage ln
beaker. pour back and forLh
alLernauve: beaker ln
ulLrasonlcaLor (3mlns)
1o decarbonaLe Lhe soda

100mL cola bev ln 23mL vol
ask. dlluLe Lo mark w/
meLhanol:waLer (2:8)

Measure caelne ln soda
Same solvenL sysLem
lollow sLeps ln A.

Aer experlmenL, wash
column w/ 30mL solvenL
(noL ad[usLed Lo pP-3.3

washlng sLep"
Results and Discussion
Chromatogram
Peak Area
Area under each single

response
Can be correlated to the

concentration of the
sample
Retention time
Distance of the peak

maxima from the
injection point
expressed in time units
Serve as identier for

given analyte on that
particular system
Most easily measurable

parameter
!
"#
Red time/Void time (t
0
)
Retention of non-retained analyte
Part of the total analyte retention time that

the analyte spends in the mobile phase
moving through the column
Serves as correction factor and allows

validation of ow rate
i.e. thiourea, uracil, and NaNO

3
Retention Factor (k)
Or capacity factor
Dimensionless, and independent on mobile

phase ow rate and column dimensions
For reproducibility, characteristic of a

chromatographic system
Retention Factor (k)
Ratio of reduced retention time to void time

Selectivity factor ()
Ability of chromatographic system to

discriminate different analytes
Ratio of retention factors
Primarily dependent on the nature of the

analytes and their interaction with the
stationary phase
Resolution
Ratio of distance between two peaks to the

average width of these peaks at baseline
Encompasses both efciency and selectivity
Has to be 1.5 to be completely separated

Efciency: Plate theory
Column is considered to be divided into a

number of hypothetical plates
Each plate has nite height (height of

effective theoretical plate, HETP) and the
analyte spends a nite time in this plate
Smaller plate height or greater number of

plates, more efcient separation
Efciency: Plate theory
Measure of chromatographic band broadening

Sensitivity
Limits of detection
lowest quantity of a substance that can

be distinguished from the absence of that
substance within a stated condence limit
Limits of quantication
concentration at which quantitative

results can be reported with a high
Calibration
External Standard Calibration
injection of a series of standards with

known concentration
peak area vs concentration
samples must have no variation in

injection volume
Calibration
Internal Standard Calibration
internal standard as an additional

component
xed volume added to all samples

(eliminated variations with injection
volume)
must be eluted with a suitable gap from

Calibration
Standards must
have similar in analytical behaviour
not expected to be found in the samples
soluble in the same solvent/eluent
not a degradation product of the sample
not affected by target analytes, surrogates, or by

matrix interferences
Calibration
not as useful for GC and HPLC methods with

non-MS detectors, unless the internal
standards could be separated from target
compounds chromatographically
for determination of caffeine, some internal

Calculation of
Standards
Peak Area vs
Concentration
Concentration of
Samples
Conclusions
In this experiment, concentration levels of

caffeine in tea, coffee, and cola beverages
were determined quantitatively by HPLC
Obtain chromatogram: x-axis (retention

time) and y-axis (intensity of the response)
Peaks observed were the result of the

sample run where the size of the peak is
proportional to the concentration of the
0.1892 M and 0.1587 M are the caffeine

concentration detected for Great Taste,
0.008933 M and 0.009751 M for Lipton,
0.003162 M and 0.003198 M for Pop, and
0.005174 M and 0.0005273 M for Zesto
Average values for both calculated values

result a caffeine concentration of 0.174 M
for Great Taste, 9.342 mM for Lipton, 3.18
mM for Pop, and 5.491 mM for Zesto
END

High Performance Liquid Chromatography: Barba, Biadomang, Dulos CHEM 127.1

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High Performance

Before: large columns, large particles, under

Giddings Prediction: smaller particles, under

Csaba Horvath and

built rst HPLC

In LC, rate of solute distribution between

to minimize diffusion and time required for

smaller eddy diffusion (small A value)

B is usually small for

A is usually small and

HPLC microparticles: high-purity silica

low in trace metal content

pore sizes: 60 to 100 angstroms, 300 for

It can be partition (liquid-liquid) or

e.g. silica gel

forces the mobile phase

introduces liquid sample

nonpolar S.P. polar M.P.

S.P: aromatic or heterocycles to provide to

polar S.P. nonpolar M.P.

RI: measure of molecules ability to

HPLC can be used for quantitative and qualitative

preparation of pure compounds

Area under each single

Can be correlated to the

Distance of the peak

Serve as identier for

Most easily measurable

Retention of non-retained analyte

Part of the total analyte retention time that

Serves as correction factor and allows

i.e. thiourea, uracil, and NaNO

Dimensionless, and independent on mobile

For reproducibility, characteristic of a

Ratio of reduced retention time to void time

Ability of chromatographic system to

Ratio of retention factors

Primarily dependent on the nature of the

Ratio of distance between two peaks to the

Encompasses both efciency and selectivity

Has to be 1.5 to be completely separated

Column is considered to be divided into a

Each plate has nite height (height of

Smaller plate height or greater number of

Measure of chromatographic band broadening

lowest quantity of a substance that can

concentration at which quantitative

External Standard Calibration

injection of a series of standards with

peak area vs concentration

samples must have no variation in

Internal Standard Calibration

internal standard as an additional

xed volume added to all samples

must be eluted with a suitable gap from

Internal Standard Calibration

have similar in analytical behaviour

not expected to be found in the samples

soluble in the same solvent/eluent

not a degradation product of the sample

not affected by target analytes, surrogates, or by

Internal Standard Calibration