HPLC Detectors Handouts

Detection in HPLC
Selecting the Right Detector:

Types of Detectors in HPLC
The Detector is the Eye

Eye of the HPLC System
1. Fucose
2. Galactosamine
3. Glucosamine
4. Galactose
5. Glucose
6. Mannose
300
Detector
mV
4
6
5
UV/VIS
Refractive index
Fluorescence
Electrochemical
Conductivity
Mass-spectrometric (LC/MS)
Evaporative light scattering
Shulamit Levin
Medtechnica
Control &
Data
Processing
Appendix:
Cutoff of solvents UV
Troubleshooting of RI detector as
an example
0.00
5.00
Minutes
20.00
Waste
Fraction
Collector
a b
cd
Auto
Sampler
Pump
levins@medtechnica.co.il
shulal@zahav.net.il
http://www.forumsci.co.il/HPLC
flows 50-5000L/min)
HPLC Column
in Oven
Optical Bench of UVUV-VIS Detector
Detectors
Optical Light Path

Taper-Cell
Flow
Cell
UV/VIS
Refractive index
Fluorescence
Electrochemical
Conductivity
MassMass-spectrometric (LC/MS)
Beam Splitter
Mirrors
Sample side
Beam-Defining
Apparatus
Illumination
Lens
Deuterium
Arc Lamp
Dual
Photodiode
Reference side
Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC
Aperture
Slit
Rotating
Diffraction
Grating
190 to 600nm
Detection in HPLC
UV Chromophores
Beer's Law
Absorbance = Extinction Coefficient x Pathlength x Concentration
Reduce Pathlength
Reduce Concentration
UV Chromophores
UVUV-Vis chromophores
max
Adenine
Guanine
Cytosine
Thymine
Uracil
NADH
NAD
260.5
275
267
264.5
259.5
340
260
Em x 10-3 @ max
E = 13.4
E = 8.1
E = 6.1
E = 7.9
E = 8.2
E = 6.23
E = 18
Detection in HPLC
UV spectrum of 10 nM mobile phase
1.00
0.90
0.80
0.70
Acetic Acid
0.60
AU
0.60
AU
Remember Solvents chosen can affect detection!!
0.80
TFA
0.70
0.50
0.40
0.30
0.30
0.20
0.20
0.10
0.10
AU
0.00
0.00
210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

nm
210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

nm
pH 2.78
pH 0.94
1.00
1.00
0.90
0.90
0.80
0.80
0.70
Formic Acid
Sodium Phosphate
0.70
0.60
AU
0.60
AU
Solvent
0.50
0.40
0.50
U.V. CutCut-offs for some Common Solvents
1.00
0.90
205.2
0.40
0.50
0.40
0.30
0.30
0.20
0.20
UV Cutoff
Water
Methanol
N-Propanol
Acetonitrile
THF
Acetone
Methyl acetate
Ethyl Acetate
Nitromethane
180
205
205
190
225
330
260
260
380
Solvent
N-Heptane
Cyclohexane
Carbon tetrachloride
Chloroform
Benzene
Toluene
Methylene chloride
Tetrachloroethylene
1,2-Dichloroethane
0.10
0.10
0.00
265.1
0.00
210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

nm
295.8
210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

nm
pH 2.26
All wavelengths reported in nm.
pH 2.0
Diode Array Detector
UV Detection of AccQAccQ-Tag Amino Acid Derivatives
Tryptophan
Lysine
0.002
Phenylalanine
Isoleucine
Asparagine
Glycine
Glutamine
Ornithine
Leucine
0.004
Serine
0.006
Glutamic Acid
0.010
0.008
Hydroxyproline
0.012
Aspartic Acid
AU 0.014
Tyrosine
Cysteic Acid
Vaine
Mtehionine
0.018
0.016
NH3
AMQ
0.020
Histidine
Arginine
Threonine
Alanine
0.022
Proline
Alpha-aminobutyric acid
0.024
0.000
15.00
20.00
25.00
30.00
35.00
Minutes
40.00
45.00
50.00
55.00
UV Cutoff
197
200
265
245
280
285
232
280
225
Detection in HPLC
Extraction of 3D Data
PDA Spectrum Index Plot
DNPH Derivatives 0.25 ng Each Peak
Absorbance
Chromatogram
Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm
1
nm
440.00
420.00
420.00
400.00
400.00
380.00
360.00
380.00
360.00
340.00
340.00
320.00
320.00
300.00
300.00
280.00
280.00
260.00
260.00
Time
Spectrum
Absorbance
440.00
0.0006
0.0006
0.0004
AU
0.0004
AU
0.0002
0.0002
0.0000
0.0000
Wavelength
Coelution of 2 Peaks
Coelution detection at a
single wavelength
Peak Purity Measurement
300.00
nm
250.00
Coelution is the sum of

absorbance of 2 peaks
A and B
200.00
Spectrum at
maximum impurity
is different
0.40
Maximum
Impurity
AU
AU
Spectra at apex and

inflection points are
displayed
0.20
0.00
2.20
2.40
2.60
2.80
3.00
Detection in HPLC
Maximum Impurity Detection
Determination of Peak Purity
Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng 360nm 996PDA 360.0 nm
nm
0.00030
0.00020
440.00
420.00
400.00
380.00
360.00
340.00
320.00
300.00
280.00
260.00
Coelution of DNPH
Hexaldehyde and
2,5-Dimethylbenzaldehyde
Peak Purity
nm
S ta n d a rd
0.00030
0.00020
AU
AU
0.00010
Hexaldehyde
0.00000
0.00000
0.00010
18.60
18.80
Minutes
19.00
Time
T im e
Peak Purity analyzes all spectra (minimum

15) within a peak against the apex spectrum
of the peak itself.
19.20
Different Spectra 53 deg
Theophylline
Dyphylline
Absorbance
Absorbance
240.00
280.00
320.00
Similar spectra for

structurally related
compounds
230.00
250.00
nm
Spectral match of apex spectrum of the

unknown against the apex spectrum of a
standard, stored in a users library.
10 deg of Spectral Contrast
Ethylparaben
EthylPaba
200.00
Unknow n
0.00010
2,5-Dimethylbenzaldehyde
-0.00010
18.40
Spectral Matching
Absorbance
440.00
420.00
400.00
380.00
360.00
340.00
320.00
300.00
280.00
260.00
270.00
nm
290.00
310.00
Detection in HPLC
Spectral Contrast 0.5 Degrees
Spectral Contrast can

differentiate these
spectra
200.00
240.00
280.00
Analyte and 2 Impurities
Very similar spectra,

CH2 difference
320.00
Absorbance
Absorbance
Methylparaben
Ethylparaben
Spectra of nonnon-UV Active Compounds
210.00
nm
230.00
250.00
270.00
nm
Refractive Index Detector

Detectors
UV/VIS
Refractive index
Fluorescence
Electrochemical
Conductivity
290.00
Detection in HPLC
Differential Refractive Index Detector
Refractive Index Detector

No sample = n
S
R
LAMP
With sample = n+
LED
To Amplifier
S
R
X = Const x n
Sugar Analysis
Polymer Analysis
800.00
750.00
Dextrose
Sucrose
700.00
80.00
600.00
Lactose
550.00
20.00
500.00
MV
-20.00
400.00
350.00
-40.00
300.00
-60.00
1260000
450.00
0.00
250.00
-80.00
200.00
-100.00
150.00
-120.00
192300
100.00
-140.00
50.00
Bagel Extract
Dow 1683
0.00
-160.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
18.00
Minutes
96400
Maltose
40.00
190000
650.00
60.00
mV
10300
Fructose
100.00
2890000
120.00
5570
SampleName: GPC STDS
SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard
20.00
22.00
24.00
Minutes
26.00
28.00
30.00
Detection in HPLC
Lipids
Detectors
250 ng on column
1=Tristearin
2=Myristic acid
Styragel HR 0.5,
4.6 x 300 mm,
35C, 0.35 mL/min
Del RIU
dRI sensitivity =
32X, 32C
5.0
6.0
7.0
UV/VIS
Refractive index
Fluorescence
Electrochemical
Conductivity
8.0
Minutes
ExcitationExcitation-Emission Spectra
Fluorescence Process
Lifetime= 10-9 10-15 sec
Excitation
Energy Levels
Maximum of
Excitation Spectrum
Stokes shift
Excited States
Emission
Ground State
Maximum of
Emission Spectrum
Detection in HPLC
Fluorescence Detectors
Fluorescence Detector Optical Bench
Excitation filter
P hotom ultiplier
tube
Cell
E m ission
G rating
LAMP
M irror
E m ission
S lit
Flow
C ell
Torroidal M irror
Emission filter
B eam S plittter
E xcitation
G rating
E xc itation
S lit
P hoto
diode
Photomultiplier
Torroidal M irror
M irror
UV vs Fluorescence Sensitivity
Detectors
AccQ-Tag amino acid
analysis
AMQ
Fluorescence
Excitation=250 nm
Emission=395 nm
Response
UV 254 nm
20.00
40.00
UV/VIS
Refractive index
Fluorescence
Electrochemical
Conductivity
60.00
Minutes
Detection in HPLC
Electrochemical Detector
Electrochemical Detection of Catecholamines & Related Compounds
Reference Electrode
2
3
Working Electrode
Analyte is oxidized or reduced
1.
2.
3.
4.
5.
6.
7.
Norepinepherine
Epinepherine
Norm etanepherine
Dopam ine
M etanepherine
3-M ethoxytyram ine
4-M ethoxytyram ine
nAm ps
150 ppb
200 ppb
50 ppb
200 ppb
200 ppb
75 ppb
500 ppb
5
7
6
Electrolyte (mobile phase)

Auxiliary Electrode
0.00
As compounds are oxidized or reduced, a current proportional to concentration is produced.
4.00
2.00
6.00
8.00
M inutes
Pulsed Amperometric Detection of Monosaccharides
1.
2.
3.
4.
5.
6.
300
UV/VIS
Refractive index
Fluorescence
Electrochemical
Conductivity
mV
Fucose
Galactosamine
Glucosamine
Galactose
Glucose
Mannose
Detectors
4
6
5
0.00
5.00
10
Minutes
20.00
10.00
12.00
Detection in HPLC
Conductivity Detector
Conductivity Detector
Mobile phase
M o b ile p h a s e
Mobile phase plus sample
M o b ile p h a s e p lu s s a m p le
Anion Analysis by IC
1.
2.
3.
4.
5.
6.
7.
1.40
3
2
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
1 ppm
2 ppm
4 ppm
4 ppm
4 ppm
6 ppm
4 ppm
Column:
Eluent:
Flow rate:
Injection vol.:
Detection:
Anion analysis by IC
Waters IC-Pak Anion HC

Borate/Gluconate
2.0 mL/min
100 L
Direct Conductivity
1.60
Detection:
Direct Conductivity after

Suppression
1.20
4
5
7
0.80
S
0.40
1.05
0.00
0.05
Column:
Eluent:
11
0.00
5.00
10.00
Minutes
15.00
20.00
25.00
0.00
4.00
8.00
1 ppm
2 ppm
4 ppm
4 ppm
4 ppm
6 ppm
4 ppm
Waters IC-Pak Anion HR

1.2 mM Sodium Carbonate/
1.2 mM Sodium Bicarbonate
Flow rate:
1.0 mL/min
Injection vol.: 50 L
0.01
0.00
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Detection: UV (PDA) at 214 nm
0.04
0.03
AU
0.02
0.70
1.
2.
3.
4.
5.
6.
7.
12.00
16.00
Minutes
20.00
24.00
Detection in HPLC
Applications
Detectors
Sensitivities for compounds such as phenol, catecholamines,
nitrosamines, and organic acids are in the picomole (nanogram)
range.
UV/VIS
Refractive index
Fluorescence
Electrochemical
Conductivity
The mobile phase must be made electrically conductive, usually

by the addition of a suitable salt:
Ion Exchange
Reversed Phase and Ion-Pair RP
No normal phase separations
Typical LC/MS System Progression

ANALYZER

ANALYZER
ION DETECTOR
ION DETECTOR
+
+
SOURCE
+
+
+
+
+
+
+
+
+
+
DETECTION OF IONS
+
+
SOURCE
+
+
+
+
+
+
SORTING OF IONS
SAMPLE DESOLVATION
AND IONIZATION
+
+
+
+
SORTING OF IONS
++
+
DETECTION OF IONS
SAMPLE DESOLVATION
AND IONIZATION
LC/MS
INTERFACE
LC/MS
INTERFACE
HPLC
MASS SPECTRUM
DATA SYSTEM
HPLC
MASS SPECTRUM
DATA SYSTEM
CHROMATOGRAM
CHROMATOGRAM
12
Detection in HPLC
Transition from LC to MS
APCI Mechanism
State of Matter: Liquid to Gas
Ionization produces
solvent ions
x
Charge State: Neutral

Neutral to Ion
M
x
Pressure: 760 torr to 10-5 to 10-8 torr
Heated Nebulizer
xH+
Corona
Needle
X = Solvent Molecules e.g.H2O, MeCN

M = Sample Molecule
Electrospray Ionization
13
XH+
xH+
M
x
x
x
MH+
x
MH+
x
x
The solvent ions react

with analyte molecules
forming clusters
Positive or Negative?
Basic Compounds (-NH2)
(M+H)+
Acidic Compounds (-CO2H, -OH)
(M-H)-
xH+
Detection in HPLC
Recognizing Multiply Charged Ions
Mass Range
Multiply Charged Molecules
Horse Heart Myoglobin
Mass spectrometers operate on the basis of mass-to-charge ratio (m/z).

Mass assignments are normally made assuming a single charge per ion
(i.e. m/z = m)
n = 23, m/z = 738

n = 22
n = 21
n = 20
n = 19
Single charge
Mass = (M+H)
Double charge
Mass = 1/2 (M+2H)
n charge
Mass = 1/n (M+nH)
n = 18
n = 17
n = 16, m/z = 1060
Isotopes of doubly charged ions are separated by 0.5 Da

Acquired Mass range
Deconvolution by MaxEnt
Hemoglobin
Hemoglobin Spectrum
Presence of More Than One Charged Envelope
100
1081.60
100
Calculated Mass
15125.0
1164.52
1009.36
1261.64
%
%
1133.92
15857.0
15866.0
1376.08
1221.18
1323.14
1058.83
15149.0
0
1000
14
1050
1100
1150
1200
1250
1300
1350
m/z
1400
mass
15000 15100 15200 15300 15400 15500 15600 15700 15800 15900 16000 16100
Detection in HPLC
Multiply Charged Ions How Many Charges?
1 Da
ANALYZER
ION DETECTOR
+
+
SOURCE
(M+H)+
+
+
+
+
+
+
SORTING OF IONS
++
+
DETECTION OF IONS
SAMPLE DESOLVATION
AND IONIZATION
LC/MS
INTERFACE
0.5 Da
HPLC
DATA SYSTEM
MASS SPECTRUM
(M+2H)2+
CHROMATOGRAM
TT im
im ee O
O ff FF lig
lig hh tt M
M aa ss ss A
A nn aa ly
ly zzee rrss
Mass
Spectrometer
Spectrometers
Analyzers
FTFT-ICRICR-Spectrometer
Magnetic Fielt B
SSO
OU
UR
RC
CEE
R
RE
E FF LL E
EC
C TT R
RO
ON
N O
O FF FF
D
DR
RIF
IFTT TTU
UBBEE
D
DEETTEEC
CTTO
OR
R
LLIN
INEEA
AR
RM
MO
OD
DEE
D
DEETTEEC
CTTO
OR
R
R
REEFFLLEEC
CTTR
RO
ON
NM
MO
OD
DEE
Elektroden
Source
Sender
R
RE
E FF LL E
EC
C TT R
RO
ON
N O
ON
N
SSO
OU
UR
RC
CEE
Trapping Plates
Electrodes
D
DR
RIF
IFTT TTU
UBBEE
DC
D
DEETTEEC
CTTO
OR
R
LLIN
INEEA
AR
RM
MO
OD
DEE
ANALYZER
ION DETECTOR
D
DEETTEEC
CTTO
OR
R
R
REEFFLLEEC
CTTR
RO
ON
NM
MO
OD
DEE
Receiver Plates
Filament
DC
DC
Transferoptic
SOURCE
Ion Traps
Transmitter Plates
199
End Cap Electrode
+
+
Axial Modulation
+
+
Ring Electrode, Rf
+
+
+
+
SORTING OF IONS
++
+
+ + +
+
+ +++
+
DETECTION OF IONS
SAMPLE DESOLVATION
AND IONIZATION
Inlet
Starting with the quadrupole
LC/MS
INTERFACE
Electron Multiplier
190
Resonant Ion
HPLC
Nonresonant Ion
Detector
V(t) = - Vdc - Vrf cost
Nier-Johnson-Geometry (EB)
Slit
Electrostatic Sector
MASS SPECTRUM
Magnetic sector
(ESA)
V(t) = Vdc + Vrf cost
Detector
dc and Rf voltages
Source
15
Slit
Ion
Source
DATA SYSTEM
CHROMATOGRAM
Detection in HPLC
Mass Spectrometer 3D Run
MS Detectors
246.00
Mixture
Electron Multiplier
1: Diode Array
4.65 5.05
Abs
210.00
Int.
UV-Diode Array Chromatogram

with poor resolution
294.00
8.62
0 200210220230240250260270280290300310
nm
5.65
8.02
10.62
Mass Analyser
+ + + + + +
-
3.82
Conversion Dynode
(Voltage 1- 20 kV)
0.74
2.00
4.00
6.00
8.00
Time
10.00
Electron Multiplier
Mix
(10.696)
(voltage setting lower than

Dynode)
Total-Ion-Current MS Chromatogram
with poor resolution
Photomultiplier
Current is measured
1: Scan ES+
4.34e5
262.87
100
59.99
213.90
%
222.87
235.87
Mixture
263.87
1: Mass Chromatogram
195.98
68.92
4.65 5.05
Int.
98.85
76.87
8.62
120.80
128.82
240.88
264.85
267.91 287.01 309.02
170.92
333.84
0
60
80
100
120
140
160
180
200
220
240
260
280
300
5.65
dynode
8.02
10.62
phosphor
photomultiplier
3.82
0.74
0
2.00
(10.696)
100
8.00
XTerra MS C18, 2.1 x 50 mm ( 5 m)
213.90
222.87
235.87
2.56
263.87
68.92
98.85
128.82
76.87
Mixture
4.655.05
Int.
60
80
100
195.98
120.80
120
140
240.88
309.02
170.92
180
200
220
240
260
100
264.85
267.91 287.01
160
280
300
320
333.84
340
295
8.11e4
m/z
10.62
4.00
6.00
8.00
10.00
1.57
100
Extracted Ion Chromatogram

of a single Component from
a mixture of Components
3.82
2.00
295 4.59e4
N
(4)
280
2.21e5
2.16
100
1: Scan ES+
262.87
4.59e5
10.60
280 1.13e5
(2)
281
100
100
O
Time
Mix
264
1.26e5
100
233
1.29
260
1.53e5
100
O
OH
260
N
H
(1)
0
100
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Time
Ding
7.67e4
265
264
(3)
NH
100
16
296
8.02
100
8.62
5.65
0.74
Time
10.00
1: Scan ES+
4.34e5
262.87
59.99
6.00
LC-MS Analysis
Selectivity of Mass Spectrometer Detector

Mix
4.00
1.57
2.16
1.29
2.56
1.00
2.00
Total Ion Chromatogram

3.50e5
3.00
Time (min)
4.00
5.00
9.25e4
261
0
100 125 150 175 200 225 250 275 300
Mass/Charge (m/z)
10 L injection of 200 ng/mL sample (in 40%
MeOH),1=Propranolol, 2=Doxepin, 3=Nortriptyline,
4=Trimipramine, 65/35 0.1 % Formic Acid / MeCN
0.2 mL/min
320
340
m/z
Detection in HPLC
Typical Quantitative Analysis Using Triple Quadrupoles:
Simultaneous MRM analysis of 6 amphetamines
Triple Quadrupoles:
Quadrupoles: MSMS-MS Modes
Daughter
Daughter (Product)
(Product) Ion
Ion Spectra
Spectra
MS1
MS1
Collision
Collision
Cell
Cell
Std_017
100
MS2
MS2
Static
Static
Typically
Typically used
used in
in Quantitative
Quantitative Work
Work of
of
Scanning
Scanning
Triple
Triple Quadrupoles
Quadrupoles
Parent
Parent (Precursor)
(Precursor) Ion
Ion Spectra
Spectra
MS1
MS1
Collision
Collision
Cell
Cell
MS1
MS1
Collision
Collision
MS2
MS2
0
Std_017
100
%
MS2
MS2
MDEA
0
Std_017
100
Static
Static
Static
Static
Constant
Constant Neutral
Neutral Loss
Loss Spectra
Spectra
MS1
MS1
Collision
Collision
Cell
Cell
Static
Static
MRM of 12 Channels ES+

165.85 > 148
2.05e7
2.25
Ephedrine
0
Std_017
100

179.8 > 163
4.13e6
3.20
MDA
%
0
Std_017
100
MS2
MS2

135.8 > 91
4.29e6
2.95
Amphetamine
%
Scanning
Scanning

149.95 > 91
1.49e7
3.80
Methamphetamine
0
Std_017
100
Cell
Cell

208 > 162.9
1.73e7
5.10
Multiple
Multiple Reaction
Reaction Monitoring
Monitoring
0.50
1.00
1.50
2.00

193.65 > 162.7
6.48e6
3.95
MDMA
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Time
Scanning
Scanning
Scanning
Scanning
Highly specific and sensitive chromatograms
Evaporative Light Scattering - ELS

Detectors
Lamp
UV/VIS
Refractive index
Fluorescence
Electrochemical
Conductivity
To detector cell
Detection
Nebulizer
Nebulization
17
Desolvation
Detection in HPLC
Scattering Models
Rayleigh Scattering Why the Sky is blue
I = I0 8 4 N2 (1 +cos2)
Scattering is dependent on particle size D Increasing

particle size
4R2
Scattering is independent of the particles chemical properties, where:
N = # of particles
= Polarizability i.e. the sum of the dipoles of all the molecules in the particle.
For a homogeneous particle this is proportional to the particle volume.
R = Distance of observer from scatterer
Dependence on wavelength of incident light, shorter wavelengths produce
greater scattering
If D/< 0.1
then I = f (D6)
0.1<D/< 10
then I = f (D4)
D/>10
then I = f (D2)
D C1/3
(Often see solute density)
I (Cb)
With 2>b>2/3
2 is the limiting value for Rayleigh
symmetrical scattering
I = Intensity of the scattered light

= wavelength of the light
Depends on which type of scattering is

predominant
Non-linear mass detector
use chromatography data software
quadratic curve or log/log curve to fit
calibration curve
ELSD vs UV
18
ELSD vs RI
Detection in HPLC
ELSD Used with Other Detectors
Not a Universal Detector

Typically Used with Other Detectors
PDA to monitor UV/Vis

friendly compounds
Diode
arrayTIC
TIC
Diode Array
UV TIC
Mass Spec to verify that
compound has been
synthesized
ES+TIC
MS ES+ TIC
ELSD to monitor all
compounds and determine
purity levels
ELSD
ELSD
2
4.5
Min
See NonNon-UV Absorbing Compounds
See Your Peaks Faster

Use of Gradients Versus Isocratic
ELSD
RI Detection
Diode Array
TIC
ELSD Detection
1
Min
Erythromycins Separation
19
Detection in HPLC
Evaporative LightLight-scattering Detector
90.00
80.00
Estriol - 1.210
100.00
Estradiol - 0.849
110.00
NaCl - 0.207
Estrogen analogues and salt
70.00
mV
60.00
50.00
40.00
30.00
20.00
10.00
0.00
0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00
Minutes
20

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Detection in HPLC

Selecting the Right Detector:

The Detector is the Eye

Optical Bench of UVUV-VIS Detector

Optical Light Path

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Remember Solvents chosen can affect detection!!

210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

U.V. CutCut-offs for some Common Solvents

210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

210.00 220.00 230.00 240.00 250.00 260.00 270.00 280.00 290.00

All wavelengths reported in nm.

Diode Array Detector

UV Detection of AccQAccQ-Tag Amino Acid Derivatives

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Millennium PDA Spectrum Index Plot - SampleWeight 0.25 ng - PDA 360.0 nm

Peak Purity Measurement

Coelution is the sum of

Spectra at apex and

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Determination of Peak Purity

Peak Purity analyzes all spectra (minimum

Different Spectra 53 deg

Similar spectra for

Spectral match of apex spectrum of the

10 deg of Spectral Contrast

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Spectral Contrast can

Analyte and 2 Impurities

Very similar spectra,

Spectra of nonnon-UV Active Compounds

Refractive Index Detector

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Refractive Index Detector

SampleName: GPC STDS

SampleName: Sugars D Vial: 1 Inj: 1 Ch: SATIN Type: Standard

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Lifetime= 10-9 10-15 sec

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Fluorescence Detector Optical Bench

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Electrochemical Detection of Catecholamines & Related Compounds

Electrolyte (mobile phase)

As compounds are oxidized or reduced, a current proportional to concentration is produced.

Pulsed Amperometric Detection of Monosaccharides

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

Mobile phase plus sample

Waters IC-Pak Anion HC

Direct Conductivity after

Waters IC-Pak Anion HR

Detection: UV (PDA) at 214 nm

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

The mobile phase must be made electrically conductive, usually

Typical LC/MS System Progression

Typical LC/MS System Progression

Dr. Shulamit Levin, Medtechnica- www.forumsci.co.il/HPLC

State of Matter: Liquid to Gas

Charge State: Neutral

Pressure: 760 torr to 10-5 to 10-8 torr

X = Solvent Molecules e.g.H2O, MeCN

The solvent ions react