Detection in HPLC - Qualification

Qualification of Detectors
Properties of Detectors

•Selectivity SELECTIVITY
•Sensitivity and detection limit
•Stability
•Linear range
•Dynamic Range
•Reproducibility SPECIFIC UNIVERSAL
•Effect on peak shape
•Maintenance

A selective detector allows one to see only components of

interest despite of their co-elution with any others.

Sensitivity Linear Dynamic Range (LDR)

R
E • Linear range is based upon the following equation:

S R=SC + N
R: Detector response
P S: Sensitivity
O C: Concentration
N: Noise Signal (intercept)
N
• For linear detector the equation becomes R=RC,
S response is linear from the limit of detection (LOD)
LOD LOQ
E to the upper concentration level which produces a
deviation from linearity of about 5%.
LOD = 3 x N
CONCENTRATION LOQ = 10 x N

Sensitivity of a detector is not the minimum

amount that can be detected.

Dr. Shulamit Levin, Medtechnica

 Detection in HPLC - Qualification
Two different Flow Cell Sensitivity - Overlay Signal to Noise
0.24

hsignal = 2 x
0.22

hnoise
High Sensitivity
0.20

0.18

0.16

0.14

0.12

0.10
AU

hsignal
0.08

0.06 Analytical
0.04

0.02

0.00

-0.02

-0.04
hnoise
-0.06
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Minutes

Importance of Sensitivity: Impurity Profile

LOD & LOQ Retention
Signal-to-
Compound Time Area %
Noise
(Minutes)
Impurity 1 2.174 0.0054 4.1
Impurity 2 2.231 0.0229 16.4
Limit of detection Lowest concentration that can be Butamben 2.263 99.954 64544

detected Signal-to-noise ratio of 2:1 or 3:1 Impurity 3 2.504 0.0037 2.6

Impurity 4 2.549 0.0106 5.8
Impurity 5 2.714 0.0034 2.2

Limit of quantitation Lowest concentration that can

be determined with acceptable precision. Signal-to-
noise ratio of 10:1

Dr. Shulamit Levin, Medtechnica

 Detection in HPLC - Qualification
Increase Signal-
Signal-to-
to-Noise Ratio Detector Noise

Signal-to-noise (S/N)
is peak height to
6:1 noise

Increase S/N by
3:1 8:1 increasing peak
height

Increase S/N by
decreasing noise

Flow Cell Comparison - Noise HPLC System 1

Ave baseline noise from 48 – 58min at 95%A / 5%B = 0.906

0.0003 Threshold = 154

0.0002
Current TUV
0.0001

0.0000

-0.0001

-0.0002

-0.0003
Analytical Flow Cell
AU

-0.0004
Peak Width = 24
Enhanced TUV
-0.0005

-0.0006
Noise
-0.0007
Comparison @
-0.0008
230 nm High Sensitivity Flow Cell
-0.0009
Enhanced TUV Propiophenone peak
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 Mean R.T. = 15.247 Mean Area = 1,014,539 Mean Signal-to-Noise = 177
Minutes
Std. Dev. = 0.003 Std. Dev. = 4,248 Std. Dev. = 21.6
% RSD = 0.02 % RSD = 0.419 % RSD= 12.2

Dr. Shulamit Levin, Medtechnica

 Detection in HPLC - Qualification
HPLC System 2
Ave baseline noise from 48 – 58min at 95%A / 5%B = 0.779
Baseline Noise
0.002
5 ng of the five peptide test mixture on column can be easily distin guished from the
Threshold = 188 baseline in the chromatograms generated on a good HPLC.
0.001

AU
0.000

-0.001
HPLC 3
-0.002
48.00 50.00 52.00 54.00 56.00 58.00
Minutes

0.40

Peak Width = 24
HPLC 2

propiophenone - 14.583
0.30

0.20
AU

0.10

0.00

-0.10
0.00 5.00 10.00 15.00 20.00 25.00 30.00 HPLC 1
Minutes
Propiophenone peak
Mean R.T. = 14.580 Mean Area = 1,046,355 Mean Signal-to-Noise = 207
Std. Dev. = 0.017 Std. Dev. = 8,813 Std. Dev. = 29.5
% RSD = 0.120 % RSD = 0.84 % RSD= 14.3

Increasing Signal to Noise Chromatographic Sensitivity

Single Wavelength vs Maxplot
Factors Affecting Noise in UV
Factors Increasing UV Signal Detectors
0.010
Optics bench design
0.008 220 nm Maxplot
Increase sample concentration Lamp energy
0.006
Increase injection volume Wavelengths

AU
0.004
Choice of wavelength (s) Mobile phase composition
0.002
Low volume flow cell Pump pulsation
0.000
Flow cell pathlength Electronics
-0.002
0.0 2.0 4.0 6.0 0.0 2.0 4.0 6.0
Minutes Minutes

Dr. Shulamit Levin, Medtechnica

 Detection in HPLC - Qualification
Noise and Drift
Baseline Stability
Noise, drift, and smallest detectable peak.

SHORT
RANGE

NOISE

LONG
RANGE
DRIFT

Baseline Noise Linearity-

Linearity-Linear Dynamic Range
0.005

R
0.004

E
0.003

Max linear response

0.002
S
Analytical
0.001
P
AU

0.000 O
High Sensitivity
-0.001 N
-0.002 S
-0.003
E 2 x detector noise
-0.004

CONCENTRATION
0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80
Minutes

Dr. Shulamit Levin, Medtechnica

 Detection in HPLC - Qualification
Linearity Test Results Background Response

3.5

2.5
Absorbance (AU)

1.5

0.5

0
0 10 20 30 40 50 60 70 80
% Absorber in Eluent

Background Absorbance Dynamic Range

Reduces Dynamic Range R
E
S
P
O
N
S
E

Time
The range of solute concentration over which the detector
continues to respond to changes in solute concentration

Dr. Shulamit Levin, Medtechnica

 Detection in HPLC - Qualification
Dynamic Range Contribution to Band Broadening

RESPONSE TIME FLOW-CELL VOLUME

Flow Cell Comparison - Sensitivity

Time constant
Analytic High 0.24

0.22

al Sensitivity Delta 0.20

0.20 0.18

Area 140955 326881 2.319 0.16

0.14

0.12

0.18 Height 93942 211138 2.248 0.10

AU

0.08

0.06

Peak Width at 0.04

0.16 0.02

13.4% 0.040497 0.042033 1.038 0.00

-0.02

-0.04

0.14 -0.06
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
Minutes

0.12
AU

0.10

0.08

0.06

0.04

0.02

0.00

4.40 4.45 4.50 4.55 4.60 4.65 4.70 4.75 4.80 4.85 4.90
Minutes

Dr. Shulamit Levin, Medtechnica

 Detection in HPLC - Qualification

Maintenance Wavelength Accuracy Test

• Flow Cell & Optics– UV-VIS, Fluorescence, RI

• Electrode – Conductivity and Electrochemical
• Nebulizer – ELS and MS

Dr. Shulamit Levin, Medtechnica