Tandem Sugar Columns: Empowering Superior Separation Performance

Everyone is familiar with sugar, as it is an important component of most foods and a significant indicator of their nutritional value. In food processing, fructose, glucose, sucrose, and maltose are often added as sweeteners. Additionally, some sugar alcohols like mannitol and xylitol play important roles in the pharmaceutical field. To ensure the safety of food and pharmaceuticals and meet modern industrial demands for the analysis and detection of sugar substances, sugar analysis chromatography columns have been widely used.

Chromatography columns commonly used for sugar analysis include amino columns with a silica gel matrix and sulfonated columns with a polymer matrix. The columns typically referred to as "sugar columns" are the sulfonated columns with a polymer matrix, such as Welch Xtimate® Sugar-H columns and Sugar-Ca columns.

Today, I’d like to share some tips discovered from using sugar columns in various cases: when a single sugar column does not achieve the desired separation in sample analysis, and adjusting the column temperature and flow rate does not result in significant improvement, you can achieve better separation by connecting two columns in series. Without further ado, let's take a look at some case studies!

Application Case 1: Sialic Acid Fermentation Broth

Liquid Chromatography Conditions

Column : Xtimate® Sugar-H (7.8×300mm, 8μm) / Xtimate® Sugar-H (7.8×150mm, 8μm)
Mobile Phase : 25mmol/L sulfuric acid aqueous solution
Column Temperature : 60℃
Detector : Refractive index detector
Detector Temperature : 40℃
Flow Rate : 0.4mL/min
Elution Time : 40 minutes
Injection Volume : 20μL

Preparation of Standard Solutions

Sialic Acid Standard Solution : Weigh 20mg of sialic acid into a 10mL volumetric flask, dissolve in water, and dilute to the mark, then mix well.
MCNNAC Standard Solution : Weigh 20mg of the standard into a 10mL volumetric flask, dissolve in water, and dilute to the mark, then mix well.
GLCNAC Standard Solution : Weigh 20mg of the standard into a 10mL volumetric flask, dissolve in water, and dilute to the mark, then mix well.
Mixed Solution Preparation : Take 1mL of each standard solution, mix well.

Chromatogram of the Mixed Solution Using a Single Xtimate® Sugar-H (7.8×300mm, 8μm) Column

	Retention time (min）	Peak area	Area	Area%	Number of theoretical plates	Tailing Factor	Resolution
1	Sialic acid	10.06	4909956	20.56	5501	0.64	-
2	M-NAC	16.07	9096636	38.10	12167	-	10.7
3	G-NAC	16.73	9869595	41.34	11687	-	1.1
	Sum		28876186	100.00

Chromatogram of the Mixed Solution Using Two Xtimate® Sugar-H Columns in Series (7.8×300mm, 8μm / 7.8×150mm, 8μm)

	Retention time (min）	Peak area	Area	Area%	Number of theoretical plates	Tailing Factor	Resolution
1	Sialic acid	14.46	5232986	21.91	5283	0.66	-
2	M-NAC	23.67	8797475	36.84	20858	1.16	13.3
3	G-NAC	24.65	9851514	41.25	20068	1.20	1.5
	Sum		23881975	100.00

Conclusion

For the separation of the last two target compounds in the chromatogram, the separation achieved using the tandem sugar columns (resolution of 1.5) is superior to that with a single sugar column (resolution of 1.1), meeting the detection requirements.

At this point, you might be concerned about the pressure increase with two columns in series. Can the instrument handle it?

Under the chromatographic conditions in this case, the pressure for a single sugar column is 1.2 MPa, while the pressure for two columns in series is only 2.5 MPa. This is not a problem for the chromatograph (most HPLC systems can withstand up to 40 MPa, although some manufacturers may set a limit at 20 MPa). Although the pressure tolerance of the sugar columns themselves is lower than that of the instrument, they can withstand up to 14 MPa.

When using other specifications of chromatography columns in series, be sure to pay attention to the column pressure!

Application Case 2: Electrolyte Injection Solution

Liquid Chromatography Conditions

Column : Xtimate® Sugar-Ca (7.8×300mm, 8μm)
Mobile Phase : Pure water
Column Temperature : 80℃
Detector : Refractive index detector
Detector Temperature : 40℃
Flow Rate : 0.5 mL/min
Elution Time : 40 minutes
Injection Volume : 10μL

Chromatogram of the System Suitability Solution Using a Single Xtimate® Sugar-Ca (7.8×300mm, 8μm) Column

	Retention time (min）	Peak area	Area	Area%	Number of theoretical plates	Tailing Factor	Resolution
1	Maltose	13.32	6764	0.38	11520	-	-
2	Maltotriose	13.92	11793	0.66	11187	-	1.2
3	Glucose	15.36	1762003	98.50	12518	1.08	2.7
4	Fructose	17.35	8362	0.47	12518	1.08	3.8
	Sum		1788922	100.00

Solution profile of two Xtimate® Sugar-Ca (7.8×300mm, 8μm) series analysis systems

	Retention time (min）	Peak area	Area	Area%	Number of theoretical plates	Tailing Factor	Resolution
1	Maltose	25.60	12779	0.36	25828	1.01	-
2	Maltotriose	26.86	21296	0.59	23441	1.09	1.9
3	Glucose	29.85	353866666	98.58	21968	1.08	4.0
4	Fructose	34.33	16829	0.47	27632	1.06	5.5
	Sum		3589569	100.00

Conclusion

For the separation of maltose and maltotriose in the system suitability solution, the resolution improved from 1.2 to 1.9 when using tandem sugar columns compared to a single sugar column.

Small Guide on Sugar Columns

Matrix: Sulfonated cross-linked polystyrene-divinylbenzene polymer

Counterions: H^⁺ and Ca2^⁺

Type: Strong cation exchange column

Features and Applications:

H-type Sugar Column:
- Acid and high-temperature resistant
- Commonly used for the separation of organic acids and sugars
- Specially designed for ribavirin analysis as specified in the Chinese Pharmacopoeia
Ca-type Sugar Column:
- Also high-temperature resistant
- Typically uses pure water as the mobile phase
- Primarily used for the separation of monosaccharides, polysaccharides, and polyols
- Designated for mannitol analysis in the Chinese Pharmacopoeia