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

High Performance Liquid
Chromatography
Barba, Biadomang, Dulos
CHEM 127.1
Monday, September 12, 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 increased flow
pressure=high plate numbers
HPLC
Csaba Horvath and
colleague S.R. Lipsky
built first 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)
more rapid mass transport between the phases, i.e. high flow
rates (small C 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
liquid S.P: coated on particles or chemically bound
Stationary phase
microporous
10 micrometers
permeable to solvent
e.g. silica gel
perfusion
12 micrometers
larger molecules
nonporous
1.5 to 2.5 micrometers
for complex peptides
ion exchange
chromatography
monolithic columns
polymers
zirconia
Components
Pump
forces the mobile phase
specific flow rate
isochratic or gradient
Injector
introduces liquid sample
autosampler
Column
Stationary phase
Detector
detect eluted
components
chromatogram
Computer
Pump
Isocratic
preparative
Gradient
analysis
Pump
Types of HPLC
Reversed phase
nonpolar S.P. polar M.P.
S.P: aromatic or heterocycles
to provide pi to pi interactions
dispersive forces
Normal phase
polar S.P. nonpolar M.P.
polar forces
Ion exchange
based on charge and size
ionic forces
Size exclusion
based on size
Detection in HPLC
Spectroscopic
UV absorption
MS detection
Refractive Index
RI: measure of molecules
ability to deflect light
amount of deflection is
proportional to
concentration
Fluorescence
only substance that
fluoresce
Detection in HPLC
HPLC
HPLC can be used for quantitative and qualitative analysis
preparation of pure compounds
trace analysis
HPLC
Examples of HPLC instruments
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
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 renLenuon 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 Tea
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 identifier for given
analyte on that particular
system
Most easily measurable
parameter
Retention volume (V
r
)
!
"#
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 flow rate
i.e. thiourea, uracil, and NaNO
3
Retention Factor (k)
Or capacity factor
Dimensionless, and independent on mobile phase flow 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
Has to be >1
Resolution
Ratio of distance between two peaks to the average width of
these peaks at baseline
Encompasses both efficiency and selectivity
Has to be 1.5 to be completely separated
Efficiency: Plate theory
Column is considered to be divided into a number of
hypothetical plates
Each plate has finite height (height of effective theoretical plate,
HETP) and the analyte spends a finite time in this plate
Smaller plate height or greater number of plates, more efficient
separation
Efficiency: 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 confidence limit
Limits of quantification
concentration at which quantitative results can be reported
with a high degree of confidence
Signal to noise ratio: LOD 3, LOQ 10
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
fixed volume added to all samples (eliminated variations with
injection volume)
must be eluted with a suitable gap from the analyte
PA
sample
/PA
IS
vs C
sample
/C
IS
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 standards used
are salicylic acid and nicotinic acid
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 analyte
response factor - concentration/area
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

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Ratio of reduced retention time to void time

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