We found that mobile phases that provided good chromatographic peak morphologies for ATP and ADP severely suppressed ionization in the ion source, and mobile phases that did not suppress ionization gave poor peak shapes for ATP and ADP (i.e., severe tailing). Therefore, to address the basal cellular energy charges [(ATP+ 1/2ADP)/(ATP+ADP+5′-AMP)] for neurons, astrocytes, and microglia, we developed a high pressure liquid chromatography-ultraviolet absorbance (HPLC-UV) assay. In this regard, aliquots of cell extracts were treated with 1-butanol, and the aqueous phase was collected, dried, and reconstituted in 0.15 M KH2PO4 (pH 6.2). Analyses of ATP, ADP and 5′-AMP were performed by HPLC with a C-18 reverse phase column (Agilent Prep-C18 Scalar, 5 μm, 100 × 4.6 mm) protected by a guard cartridge in gradient mode [buffer A: 0.15 M KH2PO4 (pH 6.2) in water; buffer B: 0.15 M KH2PO4 (pH 6.2) in 15% acetonitrile; linear gradient (%B): at 0 minutes 3.0%; from 0 to 9 minutes to 9.0%; from 9 to 25 minutes to 100.0%; from 25 to 30 minutes, 100.0%; from 30 to 31 minutes to 3.0%; from 31 to 35 minutes, 3.0%. The flow rate was 0.8 ml/minute. Adenine nucleotides in the eluate were monitored with diode array detector at 259 nm. The HPLC system was a HP Agilent Technologies 1100 series HPLC chromatograph equipped with a diode array detector (model G1315B) and autosampler (model G1313A).
1100 series hplc chromatograph
Manufactured by Agilent Technologies
The 1100 series HPLC chromatograph is a high-performance liquid chromatography instrument designed for analytical and preparative applications. It features a modular design, allowing for customization to specific user requirements. The core function of the 1100 series HPLC chromatograph is to separate, identify, and quantify components in a liquid mixture.
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Lab products found in correlation
3 protocols using 1100 series hplc chromatograph
1
HPLC-UV Assay for Cellular Energy Charges
2
HPLC Analysis of Purine Metabolites
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Hypoxanthine, xanthine, 8-aminoxanthine, 8-aminohypoxanthine, 8-aminoinosine, and guanine were measured by HPLC analysis using an Agilent (Santa Clara, CA) HPLC system, which included a HP Agilent Technologies 1100 series HPLC chromatograph equipped with a diode array detector (model G1315B) and autosampler (model G1313A ALS). Samples were heated to 90°C for 1.5 minutes (to inactivate enzymes), vortexed, and centrifuged at 14,000 rpm for 25 minutes at 4°C, and the supernatants were collected and transferred into HPLC vials. Aliquots of samples (2–15 μl) were injected onto a C-18 reverse phase column (Agilent Eclipse Plus C18, 5 μm, 4.6 × 250 mm), which was protected by a guard cartridge. Analysis was conducted in gradient mode [buffer A: 0.15 M KH2PO4 in water (pH 6.0); buffer B: 0.15 M KH2PO4 in 15% acetonitrile (pH 6.0); and linear gradient (percent B): at 0 minutes, 3.0%; from 0 to 9 minutes, 3.0%; from 9 to 25 minutes, to 100.0%; from 25 to 30 minutes, 100.0%; from 30 to 31 minutes, to 3.0%; and from 31 to 40 minutes, 3.0%]. The flow rate was 0.8 ml/min. Purines in the eluate were monitored at 259 nm. Chromatograms were processed and stored in digital form with Agilent OpenLAB CDS software. Standard curves were generated from authentic standards.
3
Lipid Composition Analysis in Oils and Diets
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Lipid class composition (TAG, DAG, MAG and FFA) of FO, RNO, REO and RAO, as well as that of all experimental diets, were determined by size-exclusion chromatography on an Agilent 1100 series HPLC chromatograph equipped with a Refractive Index Detector (RID) set at 35Cº. Oils were melted at 55ºC prior to analysis, and a solution of approximately 10 mg of oil/ml of tetrahydrofurane was prepared. The solution was filtered through a Nylon filter (0.45 µm) and injected (20 µl loop) to the chromatograph equipped with two Styragel columns (StyragelHR 1 and Styragel HR 0.5) of 30 cm x 0.78 cm i.d., filled with a spherical styrenedivinylbenzene copolymer of 5μm particle size (Water Associates, Milford, MA, USA), connected in series and placed in an oven set at 35°C. The mobile phase consisted of tetrahydrofuran at 1 ml/min. For diets, fat was previously extracted with diethyl ether following the method 2003.05 from AOAC (2005) . Data was expressed as peak area normalitzation (in %), considering the area of the peaks corresponding to TAG, DAG, MAG and FFA.
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