Theory 4

4. Theory
VITAL SCIENTIFIC N.V.

User's Manual – Vitalab Micro Series 4-1

4 Theory

4.1 General information

The analyser carries out the measurements as follows:

Parameters are measured as spectral absorbance A (λ).Reference values are determined as a

zero-adjustment on water [A (λ) = 0] automatically at the start of a test series. Then the con-
centration is determined.

Following measuring methods are available:

ƒ Absorbance
ƒ Kinetic
blank
method with/without reagent blank, with/without linearity check, with/without sample

ƒ Standard endpoint with/without reagent blank and/or with/without sample blank

ƒ Bichromatic endpoint with/without bichromatic reagent blank and/or with/without bichromatic
sample blank

ƒ Two Point with/without reagent blank, with/without sample blank

ƒ Non-linear calibration method (curve fittings)
In the following subchapters a more detailed explanation, inclusive the corresponding equa-
tions, for each method is given.

4.2 Absorbance
The absorbance is calculated according to Lambert-Beers law:

A = ε × d × c = − log T = 2 − log T %

VITAL SCIENTIFIC N.V.

4-2 User's Manual – Vitalab Micro Series

 Theory 4

4.3 Principle of Kinetic tests

Kinetic method is mostly used for enzyme activity tests. The reaction is monitored during the
whole programmed measuring time at intervals of 0.5 seconds. The measurement is checked
for linearity. If the non-linearity is greater than the programmed limit and the delta absorbance
per minute (δAbs/min) is greater than 15 milliabsorbance per minute (mAbs/min) the analyser
issues a warning message.

The calculation of Kinetic methods is as follows:

∗
 V × 1000 
U / L = δAbs / min×  total 
 ε × d ×V 
 sample 

* Note that the second part of the formula corresponds to the factor (F). Normally this factor
can be found in the package insert of the test.

The non-linearity is calculated as follows:

δAbs / min (1) − δAbs / min ( 2 )

NonLin = × 100%
δAbs / min
VITAL SCIENTIFIC N.V.

User's Manual – Vitalab Micro Series 4-3

4 Theory

4.4 Principle of Endpoint tests

Normal endpoint reactions are measured using the endpoint function. The reaction is normally
completed before the sample/reagent mixture is aspirated and so before measurement. An
endpoint measurement is performed in 2 seconds on the analyser. Different calculations are
available:

4.4.1 Standard endpoint

Abs sample
Conc = Conc
Absstd std

For calculating the sample concentration, Conc, a factor, F, is calculated as follows:

Concstd
F=
Absstd

Thus the sample concentration, Conc, is calculated as follows:

Conc = Abs × F

4.4.2 Endpoint with reagent blank

Abs sample − Abs reag

Conc = Conc std
Abs std − Abs reag

Conc std
F (actor ) =
Abs std − Abs reag

Conc sample = ( Abs sample − Abs reag ) × F

VITAL SCIENTIFIC N.V.

4-4 User's Manual – Vitalab Micro Series

 Theory 4

4.4.3 Endpoint with reagent blank and sample blank

( Abs sample − Abs reag ) − ( Abs sampleb − Abs reagbl )

Conc = Conc std
( Abs std − Abs reag ) − ( Abs stdb − Abs reagbl )

Conc
std
F ( actor ) =
( Abs − Abs ) − ( Abs − Abs )
std reag stdb reagbl

Conc sample = (( Abs sample − Abs reag ) − ( Abs sampleb − Abs reagbl )) × F

Explanation of Abbreviations:
reag means the reagent blank with active reagent
reagbl means the reagent blank with inactive reagent

sampleb means the sample blank for a sample

stdb means the sample blank of the standard

4.4.4 Bichromatic endpoint

Abs sample ,λ1 − Abs sample ,λ 2

Conc = Conc std
Abs std ,λ1 − Abs std ,λ 2

The factor calculation and the formula for bichromatic endpoints with reagent blank and/or sam-
ple blank are identical to the standard endpoint method.
VITAL SCIENTIFIC N.V.

User's Manual – Vitalab Micro Series 4-5

4 Theory

4.5 Principle of Twopoint tests

Abs sample.t1 − Abs sample.t 0
Conc = Conc
Abs std .t1 − Abs std .t 0 std

The factor calculation and the formula for two point tests with reagent blank and/or sample
blank are identical to the standard endpoint method.

4.6 Non-linear tests

Curve fitting for non-linear tests is done using the cubic spline method. This method establishes
a curve of best fit from a series of standards. An explanation of this is given in STANDARDS
section of chapter 8 under programming.

VITAL SCIENTIFIC N.V.

4-6 User's Manual – Vitalab Micro Series