The effects of dead volume on HPLC @ Glucosamine Sulfate HPLC Analysis

The effects of dead volume on Particle Size(1- 5um) HPLC and the resulting chromatography have been well studied and documented by chemist. The effects of dead volume are critical and significant when using 1-5 um columns as the impact on band broadening greatly increases compared to analytical columns. To optimize the use of narrow bore columns, it is the top priority to reduce the potential dead volume throughout the flow path in the system and the HPLC column.

So, general when dead volume can occur at the HPLC column and instrument, our focus is on the HPLC column end fittings and frits.

Replacing the Active Inlet Valve

The active inlet valve needs to be serviced in cases where the pressure ripple is unstable and the leak test verifies problems with the active inlet valve due to internal leaking. The active inlet valve contains a cartridge that is exchanged.
1. Remove the AIV using a 14 mm wrench.
2. Change the cartridge .
3. Reinstall the AIV.

PS: Properly position the AIV cable when you reinstall the valve.

The Pressure @ HPLC

Here I would like to emphasis the pressure at HPLC system again.

General lots of chemists would say"Ok, the maximum pressure is 400 Bar or something" We do not need to pay attention to that, because when it excess its maximum pressure, the system could automatically turn off." That is Right, But for me, that is not a good and satisfying answer I want to. Yes, now I want to further to discuss the pressure @ HPLC. Yes, the maximum pressure is 400 Bar. However, that is pressure of flow pathway is not the same as the 400 Bar.
When we use H2O/MeOH to wash flow pathway to elute residual analyte or air bubbles, Always CAUTION If pressure is too high, the flow cell may be damaged or broken. When removing air bubbles, set the pump pressure limit to 150 psi (10 atm) or less. Flow cell is easy be damaged or broken when we try to use high flow rate solvents to wash. So Bypass the column, pay attention to the maximum pressure and flow rate, is a Good Lab Practices Standard.

PS: A flow cell rebuilt kit is 400$.

Why FID detector display unit is V/mV

Anal. Chem., 68 (20), 3607 -3611, 1996. 10.1021/ac960556y

Accelerating Rate Calorimeter from Swden THT

The world benchmark safety calorimeter is the Accelerating Rate Calorimeter (ARC™*). The true ARC™ is manufactured uniquely by Thermal Hazard Technology.
This sophisticated system allows fulltime-temperature-pressure data under highly adiabatic conditions. An ARC™ test simulates any potential runaway reaction safely in the laboratory.

GC Assay Xylene: o-, m- and p- isomers

Dimethylbenzene:

Separation of Para- and Meta-Xylene on Capillary GC Column

Analysed on PEG 25M and FFAP g.c. columns. using a flame ionisation detector with N,N-dimethyl-. formamide as an internal standard.
Column Temp: 90 ℃, Constant.
Inlet / Detector Temp: 250℃.
Run time: 10 min.
Chromatogram: p- isomer comes first, then m- isomer later, o-isomer last.,

p-Xylene

m-Xylene

o-Xylene

Adopted from EPA Method 624

Why Solvent Filter/Degassers Matter?

Why change another Mobile Phase or new column, Got a noisy baselines.

Why Solvent Filter/Degassers Matters? ------ is a high-efficiency in-line system that is designed to remove dissolved gases from a solvent, then the mobile phase is withdrawn from the solvent reservoir and the degassed liquid is delivered to the pump.

• Degas eluents as particulates are removed
• Eliminate spurious peaks caused by outgassing in detectors
• Eliminate pump downtime caused by air locks and particulates in check valves
• Decrease piston wear; increase column life
• Filter safety into plastic-coated or heavy-walled solvent reservoirs

If the on-line degassers work 120% well, then need to clean/replace the Solvent Filter.

However, Do you still remember how to clean that?

Oh......Now I recalled some tough experiences in 3-years-Yellow Jackets Life. At 2005, made "a big mistake"-- put the Solvent Filter in the Ultra- Sonicator with MeOH. The Agilent Solvent Filter is made of glass, then the porous structure on the glass was damaged.

What solvents to be used to flush the HPLC lines to remove contaminants?

For "Dirty Analyst" --- A**P**/ Cin** *-- At least 27 different ingredients in them.

So Question is: What solvents to be used to flush the HPLC lines to remove contaminants?

My Answer is: Prepare solvent mixture consists of IPA and water, 50% each will effectively clean contaminations in the solvent tubes, capillaries, flow cells etc… in an HPLC system. Water will clean salts and other possible contaminants and IPA will clean both polar and non polar possible contaminants. Or just bypass the column with a tubing, then Using a low flow rate of 0.5 ml/min, flush the entire HPLC system for couple of hours to remove any contaminations. Or can also keep the flushing for overnight after preparing enough solvent mixture.

Go Home have a good resting, then when come back at early morning, A new trouble-free HPLC system is ready to go.

How to select buffers for my HPLC Separations?

How to select buffers for my HPLC separations?

Buffer Selection

Buffer	pKa	pH Range	UV Cutoff (A > 0.5)
Trifluoroacetic acid (TFA)	<<>	1.5-2.5	210 nm (0.1%)
KH2PO4/Phosphoric Acid	2.12	1.1-3.1	<200>
Tri-K-Citrate/hydrochloric acid 1	3.06	2.1-4.1	230 nm (10mM)
Potassium Formate/formic acid	3.8	2.8-4.8	210 nm (10 mM)
Tri-K-Citrate/hydrochloric acid 2	4.7	3.7-5.7	230 nm (10mM)
Potassium acetate/acetic acid	4.8	3.8-5.8	210 nm (10mM)
Tri-K-Citrate/hydrochloric acid 3	5.4	4.4-6.4	230 nm (10mM)
Ammonium formate	3.8 9.2	2.8-4.8 8.2-10.2	(50 mM)
Bis-tris propane� HCI/Bis-tris propane	6.8	5.8-7.8	215 nm (10mM)
Ammonium acetate	4.8 9.2	3.8-5.8 8.2-10.2	(50 mM)
KH2PO4/ K2PO4/	7.21	6.2-8.2	<>
Tris� HCI/Tris	8.3	7.3-9.3	205 nm (10 mM)
Bis-tris propane� HCI/Bis-tris propane	9.0	8.0-10.0	225 nm (10mM)
Ammonium hydroxide./ammonia	9.2	8.2-10.2	200 nm (10mM)
Borate (H3BO3/Na2B4O7 10 H2O	9.24	8.2-10.2
Glycine� HCI/glycine	9.8	8.8-10.8
1-methylpiperidine� HCI/1-methylpiperidine	10.1	9.1-11.1	215 nm (10 mM)
Diethylamine HCI/diethylamine	10.5	9.5-11.5
Triethylamine HCI/triethylamine	11.0	10.0-12.0	<>
Pyrollidine HCI/pyrollidine	11.3	10.3-12.3

PS: 0.05 M or 0.01 M is suitable for current assay requirement.

Pesticides 1,2-ethylene dibromide (EDB) and 1,2-dibromo-3-chloropropane (DMCP)

Pesticides Analysis of 1,2-ethylene dibromide (EDB) and 1,2-dibromo-3-chloropropane (DMCP) by (Agilent 6890 Micro-ECDs) after microextraction with hexane in acc ordance with U.S. EPA method 504.

Relative standard deviation (% RSD) for the entire method was less than 7% over a concentration range greater than two orders of magnitude with method detection limits of 0.003 ug/L or lower. So Nice!

Assay Details:
Inj. Vol.: 2-mL splitless injection Inlet Split/splitless; 200 °C, pulsed splitless mode (20 psi for 1 min)
Carrier Helium: 6 psi (40 °C); 3.5 mL/min constant flow.
Column: (A) 30 m, 0.53-mm id, 0.8-mm film DB-608, an equivalent of HP-608. (B) 30 m, 0.53-mm id, 1.0-mm film RTX-1701, an equivalent of HP-PAS 1701.
Oven Temp: 40 °C (4 min); 10 °C/min to 240 °C;
Detector: 330 °C.
Makeup Gas: nitrogen, constant column and makeup flow (60 mL/min)