Issue 28, 2012 - Pure glutaraldehyde Column (II) - Identification and Detection

As a chemical widely used in many fields such as papermaking, sterilization, bioindustry and oilfield water treatment, the price of glutaraldehyde remains high
.
Driven by profits, some manufacturers or distributors add formaldehyde (price about 1/16 of glutaraldehyde) and glyoxal (price about 1/4 of glutaraldehyde)
to glutaraldehyde products.

The determination of glutaraldehyde content is usually based on the Chinese Pharmacopoeia or disinfection technical specifications, and the hydroxylamine-acid titration method
is mostly used.
This titration method is based on the chemical properties of the carbonyl group and is also considered glutaraldehyde if other aldehydes
are present in the sample.
For example, the formaldehyde content of 1 g/L and the glyoxal content of 1 g/L are equivalent to 1.
67 g/L and 1.
72 g/L glutaraldehyde content
, respectively.
As a result, the measured content will be higher than the actual glutaraldehyde content
in the sample.
In response to this situation, the Dow Chemical Microbiological Control Technology Center has investigated a number of different methods for the qualitative identification and quantitative detection of glutaraldehyde containing formaldehyde, glyoxal or other aldehydes
.

1.
Qualitative detection of formaldehyde in glutaraldehyde: acetylacetonone spectrophotometrically detects whether glutaraldehyde contains formaldehyde.

This method is based on the selective reaction of formaldehyde with Nash's reagent to generate yellow 2,6-dimethyl-3,5-diacetyl-1,4 dihydropyridine
.
This method can distinguish glutaraldehyde products
with formaldehyde content exceeding 1%.

Take 15.
0 g of ammonium acetate, 0.
300 ml of glacial acetic acid and 0.
200 ml of acetylaceton, dissolve with ultrapure water and set the volume to 100 ml
.
According to the ratio of 1:10000, quantitatively dilute 50% glutaraldehyde product with ultrapure water, quantitatively pipette 1.
00ml of sample solution, add 0.
50ml of Nash reagent, mix well, and heat the reaction at 100 °C for 5 minutes
.
After cooling to room temperature, it was compared
with standard formaldehyde and blank control.
This is shown in
Figure 1.
Compared with blank and Dow glutaraldehyde samples, formaldehyde-containing products appear significantly yellow
after reaction.

2.
Gas chromatography: Gas chromatography can be used to selectively determine the content
of glutaraldehyde in products.
According to the dilution ratio of 1:100, dilute the volumetric standard glutaraldehyde solution and sample solution, inject the standard solution and sample solution at the same time, and calculate the content
of glutaraldehyde in the sample according to the peak area.
This method can accurately determine the true content of glutaraldehyde in the product, even if other aldehydes are mixed into the product, it has no effect
on the measurement results.

Commonly used chromatographic conditions: Column: Agilent DB-5 capillary column, 30m×0.
53mm× 1.
5μm; Carrier gas: helium, 3mL/min; Injection port: 150 °C, 2μL injection volume, split ratio 30:1; Heating procedure: starting temperature 60 °C, constant temperature for 2 minutes; Heating rate 15 °C/min to 230 °C, constant temperature 2min; Hydrogen ion flame detector: 250°C, hydrogen 40mL/min, air 400mL/min, tail blowing 30mL/min
.
The resulting gas chromatogram is shown in
Figure 2.

3.
High performance liquid chromatography-DNPH pre-column derivatization method: The previous two methods can distinguish glutaraldehyde products doped with formaldehyde, but cannot determine the content
of formaldehyde, glyoxal, etc.
incorporated.
Because formaldehyde, glyoxal, etc.
are difficult to detect directly, the derivatization method is mostly used to detect their content
.
When the content of other aldehydes incorporated is high, the high performance liquid chromatography-DNPH pre-column derivatization method
can be used.
This method is based on the reaction of aldehydes and 2,4-dinitrophenylhydrazine to generate hydrazone compounds, which are then separated and detected
by high performance liquid chromatography.

Take 0.
25 g of 2,4-dinitrophenylhydrazine and 0.
750 ml of 85% phosphoric acid and dissolve it with acetonitrile to 50 ml
.
According to the ratio of 1:5000, quantitatively dilute 50% glutaraldehyde product with acetonitrile, quantitatively pipette 2.
00 milli sample solution, add 0.
80 ml of 2,4-dinitrophenylhydrazine solution, mix well, react at room temperature for 1 hour, inject and analyze, and calculate the formaldehyde content
in the sample according to the peak area.
Chromatographic conditions: Column: Agilent XDB-C18 column, 150mm× 4.
6mm×5μm, room temperature; Mobile phase: 75 water/25 acetonitrile, 1.
5 mL/min; Injection volume: 10μL; Detection wavelength: 365nm
.
High performance liquid chromatography analysis of glutaraldehyde samples with high formaldehyde content is shown in
Figure 3.

4.
High performance liquid chromatography-post-column derivatization: When the formaldehyde or other aldehydes in glutaraldehyde are very low, the pre-column derivatization method cannot analyze its content
.
In this case, it is necessary to choose the post-column derivation method
.
The principle is that formaldehyde or other aldehydes in the sample are separated by high performance liquid chromatography, and then detected
after reaction with derivatization reagents.
Using this method, formaldehyde content in glutaraldehyde is less than 7.
5ppm undetectable
.

According to the dilution ratio of 1:100, after quantitatively diluting glutaraldehyde samples, the sample was directly injected for analysis, and Nash reagent was used for derivatization reagents
.
Calculate the formaldehyde content
according to the peak area.
Chromatographic conditions: Column: Phenomenex Hypersil C1 Column, 150mm× 4.
6mm×5μm, room temperature; Mobile phase: ultrapure water, 0.
45mL/min; Injection volume: 10μL; Post-column derivatization conditions: 95 °C, Nash derivatization reagent 0.
35mL/min; Detection wavelength: 410nm
.
Dow glutaraldehyde samples were analyzed and detected under these conditions, and no formaldehyde
was detected.
See Figure 4
.