ANALYSIS OF CATION AND ANION: Determination of the Lipase with Potentiometric Method

Antacid

Determination of the Lipase with Potentiometric Method

Equipment
Potentiometer = 751 GPD Titrino / Titroprocessor
Electrodes = 6.0233.100 combined pH glass electrode; input 1
6.1110.100 T sensor

Reagents
c(NaOH) = 0.1 mol/L; D0

Sample
29.5 mL substrate emulsion
0.5 mL enzyme solution

Remarks
Pancreas lipase is determined using a TIP method: The pH is adjusted to 9.20 with a pretitration (LipPre), then the sample is addedand the pH kept at 9 during 5 minutes (LipDeter). The determination is carried out in a thermostated titration vessel (e.g. 6.1418.220) at 37 °C.

Calculations:

FIP=C70*C31/C00;1;U/mg

LipStd
Factor = calculation factor
FIP = activity of the reference standard in FIP units/mg.
C01 = declared activity of FIP standard (36.2)
C02 = 1000*concentration of titrating agent (100)
C70 = mean rate from LipDeter
LipSmpl1)
FIP = activity of the sample in FIP units/mg.
C30 = calculation factor from LipStd
C70 = mean rate from LipDeter
LipSmpSC2)
FIP = Aativity of the sample in FIP units/mg.
C31 = calculation factor from silo calculation of LipStd
C70 = mean rate from LipDeter
1): Delete the following assignments in method „LipStd“:
. in key , >silo calculations, C24=RS1 and C25=RS2
. in key , >common variables, C31=C26
2): Delete the following assignment in method „LipStd“:
. in key , >common variables, C30=MN1

Solution of Arabic gum:
Dissolve 100 g Arabic gum in 1000 mL dist. water. Centrifuge until the solution is clear (app. 10 minutes with 4000 r/min). This solution can be kept several weeks if frozen in portions.

Substrate standard solution:
Add 165 mL Arabic gum solution, 20 mL olive oil (BP 73) and 15 mLdist. water and cool it in ice to 5 °C. Homogenize with an electric mixer during 30 minutes. The temperature of the solution should never rise above 30 °C. This solution can be kept up to 14 days in a refrigerator. Always homogenize the emulsion before use.

Substrate emulsion:

Stir 100 mL substrate standard solution, 80 mL TRIS buffer solution, 20 mL sodium taurocholate and 95 mL dist. water. This solution must be prepared daily.

TRIS buffer solution:
Dissolve 60.6 mg Tris(hydroxymethyl)-amino methane (C4H11NO3) and 234.0 mg sodium chloride in dist. water and fill up to 100 mL. This solution can be kept up to 3 days in a refrigerator.

Sodium taurocholate solution:
Dissolve 4.0 g sodium taurocholate (F.I.P. controlled) in dist. Water and fill up to 50 mL.

Enzyme solvent

Dissolve 10.0 g sodium chloride, 6.06 g Tris(hydroxymethyl)-amino methane and maleic acid anhydride (C4H2O3) in 900 mL dist. water. Adjust pH to 7.0 with 4N NaOH (app. 13 mL) and fill up to 1000 mL. This solution can be kept up to 3 days in a refrigerator.

Sample preparation:
Dissolve the enzyme in the enzyme solvent. The solution should contain between 8 and 16 FIP Units per mL.

Comments to parameters:

LipDeter Time windows for rate evaluation. Monitoring of rate and temperature is ON: the rate should be kept in the range of 0.08...0.16 mL/min, the temperature in 0.1°C. LipStd, LipSmpl, LipSmpSC T may be measured with .

Literature

B. Stellmach, Bestimmungsmethoden Enzyme, Steinkopff Verlag Darmstadt, 1988, p. 263

Method documentation

Results

'fr

751 GPD Titrino 15215 751.0010

date 97-02-26 time 15:53 10

pHc(init) 9.20 STAT pH LipDeter

smpl size 0.3475 mg id#1 Sample 2

C81 rate 0.1125 +/- 0.00033 ml/min

C82 rate 0.1064 +/- 0.00036 ml/min

C83 rate 0.1020 +/- 0.00029 ml/min

C84 rate 0.0980 +/- 0.00027 ml/min

C80 m.rate 0.1047 +/- 0.00017 ml/min

stop time reached

============

'cu
751 GPD Titrino 15215 751.0010
date 97-02-26 time 15:53 10
start time 60 s STAT pH LipDeter
50.0 s/div dV=0.1 ml/div
============
'fr
751 GPD Titrino 15215 751.0010
date 97-02-26 time 15:54 10
TIP LipSmpSC
smpl size 0.3475 mg id#1 Sample 2
C70…. 0.1047
FIP 35.0 U/mg
mean( 2) +/-s s/%
FIP 35.1 0.14 U/mg 0.40
TIP terminated
============
'fm
751 GPD Titrino 15215 751.0010