Analysis of human bone alkaline phosphatase isoforms: Comparison of isoelectric focusing and ion-exchange high-performance liquid chromatography

2007 ◽  
Vol 379 (1-2) ◽  
pp. 105-112 ◽  
Author(s):  
Christopher A. Sharp ◽  
Cecilia Linder ◽  
Per Magnusson
Keyword(s):  
Alkaline Phosphatase ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Ion Exchange ◽  
Isoelectric Focusing ◽  
High Performance ◽  
Bone Alkaline Phosphatase ◽  
Human Bone

4-Nitrophenol in 4-nitrophenyl phosphate, a substrate for alkaline phosphatase, as measured by paired-ion high-performance liquid chromatography.

1977 ◽  
Vol 23 (12) ◽  
pp. 2288-2291 ◽  
Author(s):  
P H Culbreth ◽  
I W Duncan ◽  
C A Burtis
Keyword(s):  
Alkaline Phosphatase ◽  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Flow Rate ◽  
Mobile Phase ◽  
High Performance ◽  
Reversed Phase ◽  
Nitrophenyl Phosphate ◽  
Phase Column ◽  
Reversed Phase Column

Abstract We used paired-ion high-performance liquid chromatography to determine the 4-nitrophenol content of 4-nitrophenyl phosphate, a substrate for alkaline phosphatase analysis. This was done on a reversed-phase column with a mobile phase of methanol/water, 45/55 by vol, containing 3 ml of tetrabutylammonium phosphate reagent per 200 ml of solvent. At a flow rate of 1 ml/min, 4-nitrophenol was eluted at 9 min and monitored at 404 nm; 4-nitrophenyl phosphate was eluted at 5 min and could be monitored at 311 nm. Samples of 4-nitrophenyl phosphate obtained from several sources contained 0.3 to 7.8 mole of 4-nitrophenol per mole of 4-nitrophenyl phosphate.

Usefulness of Cation Exchange High Performance Liquid Chromatography as a Testing Procedure

PEDIATRICS ◽  
1989 ◽  
Vol 83 (5) ◽  
pp. 849-851
Author(s):  
Titus H. J. Huisman
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Cation Exchange ◽  
Cellulose Acetate ◽  
Isoelectric Focusing ◽  
High Performance ◽  
Testing Procedure ◽  
Cellulose Acetate Electrophoresis ◽  
Blood Samples ◽  
Testing Procedures

Testing of cord blood or newborn blood samples for hemoglobin abnormalities should include clinically important hemoglobinopathies other than sickle cell anemia (SS), such as SC, SD, SO, S-β- thalassemia (thal), EE, SE, and α-thal, and should place the quality of the testing procedures (ie, accuracy of diagnosis) above quantity (ie, number of samples tested over a given period). There is no single method available that is suitable for the identification of each of the numerous abnormalities; thus, at least two, and often more than two, procedures must be used to reach a definitive diagnosis. For this reason, blood samples collected in vacutainers with ethylenediaminetetraacetic acid as anticoagulant are preferred to those collected on filter papers. The latter approach also has the disadvantage that, under a less than optimal transport system, hemoglobin is readily modified (oxidation, glycosylation, protein-protein interaction), producting extra bands or peaks in electrophoretic or chromatographic separations that interfere with an appropriate identification of various genetically determined hemoglobin variants. In our laboratories, in which hemoglobin identification has been routine for more than 25 years, we consider the following procedures acceptable primary testing methods: starch gel electrophoresis at pH 8.9, cellulose acetate electrophoresis at pH 8.5 to 8.9, isoelectric focusing, and fast cation exchange high performance liquid chromatography (HPLC). The following five methods are excellent confirmatory testing procedures: citrate agar electrophoresis at pH 6.1, cation or anion exchange macrochromatography, isoelectric focusing, cation exchange HPLC, and immunologic procedures. Combinations of these techniques will often lead to acceptable data, and the general approach followed in our institute is given in Fig 1. Cellulose acetate electrophoresis at alkaline pH is still the primary testing procedure, and citrate agar electrophoresis at pH 6.1 and micro-HPLC procedures are the main confirmatory methods.

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