The disguised chromatograph, part II: The Amino Acid Analyzer

The history of Amino Acid Analysis (AAA) is the history of chromatography itself, just before Martin and Synge win the Nobel Prize for the Partition Chromatography (1952) Stein and More were working on Amino Acid separation using starch columns (1948). But that can be the subject of another post.

The important is that, since the 50's the formula didn't change too much. The automated analyzer is still a chromatograph with post column reaction and UV (ultra-violet) or Visible detection.

Using a mobile phase gradient to separate the mixture, these aparatus changed from glass packed tubes into automated systemns controled by computer and with much more fast, accurate and practical tools.

Today standard for AAA is ion exchange columns for LC and capillary GC of Amino acids derivatives.

Some companies as Phenomenex supply kits for sample preparation, calibrators and columns. For GC and HPLC equipments. So you can run all amino acids in a non-dedicated equipment.

Take a look how things improved in last 60 years!

Chromatograms from Stein and More experiments in the 50's, the could take days to run a single sample. 

Modern analysis using high-end equipment. in two hours you can separate more than 50 peaks.

Schematic of the automatic recording apparatus used in the chromatographic analysis of mixtures of amino acids.   Spackman, D. H., Stein, W. H., and Moore, S. (1958) Anal. Chem. 30, 1190–1206

Hitachi's Amino Acid Analyzer with tray for 200 vials



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The disguised chromatograph, part I: CHN analyzer

An important academic research tool that I rarely heard about in industry is the CHN anayser. Any Organic/Synthetic chemist know that in some applications CHN are so important as NMR or Mass Spectrometry, but what many people doesn't know is that CHN is a automatized chromatograph.

It works in following way:

The sample is heated at 1000 ºC and carbon, hydrogen and nitrogen converted to CO2, H2O and NO2, after reduction of NO2 to N2 through a cooper catalyst, the gases are separated and measured with a thermal conductivity detector.

In the end you have the final percentage of each element like: C: 45%, H: 8% and N: 47%.

Some models also detect Sulfur as SO2.

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Advertizing from 30 years ago.

Analytical Chemistry, 1981.
When I look at this, I love my Windows programs!

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The Green chromatographer, part II - Narrow bore columns

The biggest enviromental concern about liquid chromatography is the waste. The widely used reversed-phase mode uses organic solventsto modify an aquous mobile-phase (MP) and elute the analytes.

The most used solvents, Methanol and Acetonitrle are toxic to environment, and shoud be, "in theory", incinerated.

Much better than giving a proper destiny to you waste, decrease its amount is a much better approach.

The Narrow bore (>4mm id)  and Microcolumn (>1mm id) LC rely on the smaller internal diameter columns, that work with a lower flow.

Narrow bore columns can work on normal HPLC equipments while Microcolumn LC need special low volume conections.

Running samples from a 4.6mm id column to a 2mm will decrease you flow 5 times! from 1ml/min to 0.2 ml/min!

The flow is proportional to the are from column, if you decrease the internal diameter to half, the cross sectional area will decrease about four times.

Just take a look at most common column diameters and the cross sectional area:

While the 4 mm id column have an area of 12.5  mm² uses 1 ml/min of  MP, the 2 mm id column has 3,1 mm² of area and requires 0.25 ml/min of MP.

The very economic 1 mm id column with 0.78 mm² only need 0,062ml/min to run in the same linear velocity.

NOTE: I 'm considering that packing is the same as the instrumental.

Example:
Fatty acids analysis on HPLC with 2.1 mm id C18 column, 85% MeOH at 0.2 ml/min.

F.O. Silva, V. Ferraz / Talanta 68 (2006) 643–645

Good luck!!

Peaks are: 1, linoleic + myristic; 2, linoleic; 3, palmitic; 4, oleic; 5, elaidic; 6, margaric (internal standard); 7, stearic phenacyl ester.

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