Special/Multi-Wavelength Method with
WTW’s photoLab® UV-VIS Spectrophotometer
Photometric Determination of D-Glucose:
| Photometer |
WTW Spectrophotometer PhotoLab 6600 UV/VIS or PhotoLab 7600 UV/VIS |
| Test |
Enzymatic UV-Test D-Glucose (10 716 251 035) from the company BOEHRINGER MANNHEIM / R-BIOPHARM. |
| Method |
Special / Multi-Wavelength |
| Measurement |
Sample and blank value at 340 nm |
Content of this documentation:
- Part 1: Common description
- Part 2: Instruction manual
- Part 3: Methodparameter, Formula design
- Part 4: Method programming
Part 1: Common description
Within the enzymatic reaction of D-Glucose to D-Gluconat-6-Phosphate an equivalent amount of Nicotinamide-Adenin-Dinucleotide / Nicotinamide-Adenin-Dinucleotide-Phosphate (NAD+/NADP+) is reduced to NADH/NADPH. NAD/NADPH has a specific absorption for the photometrical determination at the wavelength of 340 nm.
Figure 1: Absoptionsspectrum of NAD+/NADP+ and NADH/NADPH.
Part 2: Instruction manual
Determination
Glucse from Disaccharides requires a preceding digestion.
|
| Sample |
Photometer |
|
Fill into an empty 10 mm rectangular cuvette:
|
Select Multi-Wavelength method:
Glc(f) 716 251, 10 mm
 
|
1,000 ml - Reagent 1, NADP and ATP
+ 0,100 ml - sample
+1,900 ml - double distilled water
3 min. - Reaction time |
Dilution
Setting a dilution factor. The dilution facton multiplies the result with the adjusted number.
|
|
1. Absorption measurement
Glc(f) + NADP/ATP
Measurement of D-Glucose after addition of reagent 1, sample and distilled water.
|
Place the cuvette into the cuvette shaft, the measurement starts automatically.
 
After the measurement remove the cuvette from the cuvette shaft for subsequent processing.
|
|
Fill in the same cuvette:
+ 0,020 ml Reagent 2, G6P-DH and HK
10 - 15 min. Reaction time
|
|
|
2. Absorption measurement
Glc(f) + HK
Measurement of D-Glucose after addition of reagent 2.
|
Place the cuvette into the cuvette shaft, the measurement starts automatically.
After the measurement take the cuvette out of the cuvette shaft.
|
|
Continue with: „Blank value determination“
|
| Blank value |
Photometer |
|
Fill into an empty 10 mm rectangular cuvette:
1,000 ml Reagent 1, NADP and ATP
+2,000 ml Double distilled water
3 min. Reaction time
|
 |
|
3. Absorption measurement
BV + NADP/ATP
Measurement of the blank value after addition of reagent 1 and distilled water.
|
Place the cuvette into the cuvette shaft, the measurement starts automatically.
After the measurement remove the cuvette from the cuvette shaft for subsequent processing.
|
|
Fill into the same cuvette:
+ 0,020 ml Reagent 2, G6P-DH and HK mix
10 - 15 min. Reaction time
|
|
|
4. Absorption measurement
BV + HK/G6P-DH
Measurement of the blank value after addition of reagent 2
|
Place the cuvette into the cuvette shaft, the measurement starts automatically.
|
|
The result will be displayed.
|
Part 3: Method parameter and formula design
Calculation of the concentration
Hierbei gilt:
| c |
Result |
|
| V |
Volume [ml] measurement solution |
3,020 ml |
| MG |
Molar weight of Glucose |
180,16 g/mol |
| ε |
Molare Absoption coefficient (NADPH) |
Bei 340 nm = 6,3 [L / mmol / cm] |
| d |
Layer thickness of the cuvette |
1,00 cm (10 mm) |
| v |
Sample volume [ml] |
0,100 ml |
| 1000 |
Divisior for displaying the result in g/L |
|
| ΔE |
Absorption difference of sample and blank value |
ΔE = (E2-E1)Probe - (E2-E1)Leerwert |
| [g/L] |
Dimension of the result |
|
Photometrical factor (F) and measurement range:
| Rectangular cuvette 10 mm at 340 nm |
| F = 0,864 |
 |
| Measurement range:: 0,08 – 0,5 g/L Glucose |
|
Formular design:
The order of the absorption measurement follow the schema of the producer of the test kit.
The order of the measurement of formular variables within the photometer programming follow the index of this variables in ascending sequence.
Calculation of Glucose
Absorption differences
ΔEGlc = (E2Glc – E1Glc) – (E2BlankValue – E1BlankValue)
| Measurements |
| ΔEGlc |
Difference of absorption measurements |
| E1Glc |
1. Absorption measurement of the sample |
| E2Glc |
2. Absorption measurement of the sample |
| E1BlankValue |
1. Absorption measurement of the blank value |
| E2BlankValue |
2. Absorption measurement of the blank value |
For the calculation of the differences of absorption measurements regarding the photometrical factor plus a possible measurement range expansion by diluting the sample the formula for programming the photometer can look as follows:
R = 0,864 * ((A340nm_2 - A340nm) – (A340nm_4 + A340nm_3)) * K1
Where:
| R |
Result in g/L |
| 0,864 |
Photometrical factor for the determination of Glucose in a 10 mm rectangular cuvette at the wavelength of 340 nm. |
| A340nm |
1. Formula variable, Index = 1; corresponds to: E1Glc |
| A340nm_2 |
2. Formula variable; Index = 2; corresponds to: E2Glc |
| A340nm_3 |
3. Formula variable; Index = 3; corresponds to: E1BlankValue |
| A340nm_4 |
4. Formula variable; Index = 4; corresponds to: E2BlankValue |
| K1 |
Dilution- / Multiplication-factor |
Part 4: Programming the method
Common specification of the new method can be set to:
| Value |
Input ** |
Description |
| Number * |
device dependent |
Method-list numbering, arbitrary selectable; certainly, each number can be selected only one time. |
| Name * |
Glc(f) 716 251, 10mm |
Denonimation of the methode for the method list, arbitrary selectable, „Glc(f)“ = Glucose (free); „716 251“ = order number of the producer, „10mm“ = methode for the 10 mm rectangular cuvette, max. 20 characters; |
| Version * |
1 |
arbitrary version number, max. 5 characters |
| Citation form * |
Glucose |
Naming of the result, max. 15 characters |
| Unit * |
g/L |
Dimension of the result in g/L, max. 10 characters |
| Resolution |
0.01 |
2 decimal points for displaying the result; selection from a predefined list |
| Cell |
10 mm |
Cuvette type, selection from a predefined list |
| Lower limit of measuring range * |
0.08 g/L |
Lowest realistic measurement value |
| Upper limit of measuring range* |
0.50 g/L |
Highest realistic measurement value |
| Wavelength 1 |
340 nm |
All measurements are carried out at this wavelength |
| Variable |
Naming |
Description |
| K1 * |
Dilution |
Measurement range expansion; in this case a factor for the multiplication of the result if the sample was pre-diluted; max 10 characters.
The input of this value is carried out at runtime in the beginning of the method.
(max. 10 variables)
|
| Calculation formula** |
Input of numbers, letters, variables and operators with the keypad of the photometer or an external USB-keyboard.
(more than 250 characters possible) |
| R = 0.864 * ((A340nm_2 - A340nm) - (A340nm_4 - A340nm_3)) * K1 |
Condition
** |
Boehringer Mannheim: to get results with adequate accuray absorption differences should be more than 0.100 absorption units.
((A340nm_2 - A340nm) - (A340nm_4 - A340nm_3)) > 0.1
or
R > 0.08 |
| Step |
caption |
Description
(max. 20 characters) |
| Measurement 1 * |
Glc(f) + NADP/ATP |
1. Absorption measurement, sample after addition of Nicotineamide-adenine-Dinucleotide-Phosphate (NADP) and Adenonsine-Triphosphat (ATP) reagent. |
| Measurement 2 * |
Glc(f) + HK/G6P-DH |
2. Absorption measurement, sample after addition fo Hexokinase (HK) and Glucose-6-Phosphate-dehydrogenase (G6P-DH) Absorptionsmessung, Probe nach Zugabe von Hexokinase. |
| Measurement 3 * |
BV + NADP/ATP |
3. Absorption measurement, blank value after addition of NADP and ATP. |
| Measurement 4 * |
BV + HK/G6P-DH |
4. Absortpion measurement, blank value after addition of HK and G6P-DH. |
* Adjustments and labelling are arbitrary selectable, the count of characters in limited.
The decimal separator for inputting numbers is the point ‚.‘
Here you can download the whole article as a PDF:
