Automated Amino Acid AnalysisUsing an Agilent Poroshell HPH-C18Column

Author

William Long

Agilent Technologies, Inc.

Application Note

Food Testing, Agriculture, Small Molecule Pharmaceuticals

Abstract

In this application note, an automated precolumn OPA/FMOC amino acid method,

previously developed on 3.5 and 1.8 µm Agilent ZORBAX Eclipse Plus C18 columns,

is expanded to include 2.7 µm Agilent Poroshell HPH-C18 superficially porous

columns. This column exhibits good lifetime and transferability to different column

dimensions, both of which are shown in this work. Applications of the column to

fermentation products are also shown.

2

Introduction

Superficially porous particle (SPP) technology is based onparticles with a solid core and a superficially porous shell.These particles consist of a 1.7 µm solid core with a 0.5 µmporous shell. In total, the particle size is about 2.7 µm. The2.7 µm superficially porous particles provide 40 to 50% lowerbackpressure and 80 to 90% of the efficiency of sub-2 µmtotally porous particles. The superficially porous particleshave a narrower particle size distribution than totally porousparticles. This results in a more homogeneous column bed,and reduces dispersion in the column. At the same time, thethin porous shell provides lower resistance to mass transfer.The result is minimal loss of efficiency at higher flowrates [1]. Additionally, since the columns incorporate a 2 µmfrit, they are as resistant to clogging as 3.5 and 5 µm columns.Until recently, all silica-based SPP materials possessedlimited lifetime in higher pH buffers, including phosphatebuffers. To achieve these longer lifetimes, it is necessary toprotect the base particle by either surface modification orspecial bonding modification. The surface of Agilent PoroshellHPH-C18 particles are chemically modified to form an organiclayer, resistant to silica dissolution at high pH conditions,using a proprietary process.

The continuous improvement in HPLC columns andinstrumentation presents an opportunity to improve HPLCmethods. A proven ortho-phthalaldehyde/9-fluorenylmethylchloroformate (OPA/FMOC)-derivatized amino acid methoddeveloped on HP 1090 Series HPLC systems, and laterupdated for the Agilent 1100 Series, has now evolved furthertaking advantage of the Agilent 1260 Infinity Binary LC andsuperficially porous Agilent Poroshell HPH-C18 columns [2-8].

Experimental

Preparation of HPLC mobile phaseMobile phase A contained 10 mM Na2HPO4, 10 mM Na2B4O7,pH 8.2, and 5 mM NaN3. For 1 L, weigh 1.4 g anhydrousNa2HPO4 plus 3.8 g Na2B4O7•10H2O in 1 L water plus 32 mgNaN3. Adjust to about pH 8.4 with 1.2 mL concentrated HCl,then add small drops of acid to pH 8.2. Allow stirring time forcomplete dissolution of borate crystals before adjusting pH.Filter through 0.45 µm regenerated cellulose membranes(p/n 3150-0576). Mobile phase B containsacetonitrile:methanol:water (45:45:10, v:v:v). All mobile-phasesolvents were HPLC grade. Since mobile phase A isconsumed at a faster rate than B, it is convenient to make 2 L of A for every 1 L of B.

The injection diluent was 100 mL mobile phase A, plus 0.4 mLconcentrated H3PO4 in a 100 mL bottle, stored at 4 °C.

To prepare 0.1 N HCl, add 4.2 mL concentrated HCl (36%) to a500 mL volumetric flask that is partially filled with water. Mix,and fill to the mark with water. This solution is for makingextended amino acid and internal standard stock solutions.Store at 4 °C.

Derivatization reagents (borate buffers, OPA, and FMOC) areready-made solutions supplied by Agilent. They simply needto be transferred from their container into an autosampler vial.Some precautions include:

• OPA is shipped in ampoules under inert gas to prevent oxidation. Once opened, the OPA is potent for about 7 to10 days. We recommend transferring 100 µL aliquots ofOPA to microvial inserts. Label with name and date, cap,and refrigerate. Replace the OPA autosampler microvialdaily. Each ampoule lasts 10 days.

• FMOC is stable in dry air but deteriorates in moisture. Itshould also be transferred in 100 µL aliquots to microvialinserts. Label with name and date, cap tightly, andrefrigerate. Like the OPA, an open FMOC ampouletransferred to 10 microvial inserts should last 10 days(one vial/day).

• Borate buffer can be transferred to a 1.5-mL autosamplervial without a vial insert. Replace every three days.

Preparation of amino acid standardsSolutions of 17 amino acids in five concentrations areavailable from Agilent (10 pmol/µL to 1 nmol/µL) forcalibration curves. Divide each 1 mL ampoule of standards(p/n 5061-3330 through 5061-3334) into 100 µL portions inconical vial inserts. Cap and refrigerate aliquots at 4 °C.

To make the extended amino acid (EAA) stock solution, weigh59.45 mg asparagine, 59.00 mg hydroxyproline, 65.77 mgglutamine, and 91.95 mg tryptophan into a 25 mL volumetricflask. Fill halfway with 0.1 N HCL and shake or sonicate untildissolved. Fill to mark with water for a total concentration of18 nmol/µL of each amino acid.

For the high-sensitivity EAA stock solution, take 5 mL of thisstandard-sensitivity solution and dilute with 45 mL water(1.8 nmol/µL). Solutions containing extended standards areunstable at room temperature. Keep them frozen and discardat first signs of reduced intensity.

3

For primary amino acid ISTD stock solutions, weigh 58.58 mgnorvaline into a 50 mL volumetric flask. For secondary aminoacids, weigh 44.54 mg sarcosine into the same 50 mL flask.Fill half way with 0.1 N HCl and shake or sonicate untildissolved, then fill to mark with water for a final concentrationof 10 nmol each amino acid/µL (standard sensitivity). Forhigh-sensitivity ISTD stock solution, take 5 mL ofstandard-sensitivity solution and dilute with 45 mL of water.Store at 4 °C.

Calibration curves are made using two to five standardsdepending on experimental need. Typically, 100 pmol/µL,250 pmol/µL, and 1 nmol/µL are used in a three-pointcalibration curve for standard-sensitivity analysis.

Pump parametersPump parameters for all methods include compressibility(×10–6 bar) A: 35, B: 80, with minimal stroke A, B of 20 µL.

Online derivatizationDepending on the autosampler model, the automated onlinederivatization program differs slightly. For the Agilent G1376Cwell plate automatic liquid sampler (WPALS), with injectionprogram:

1. Draw 2.5 µL from borate vial (p/n 5061-3339).

2. Draw 1.0 µL from sample vial.

3. Mix 3.5 µL in wash port five times.

4. Wait 0.2 minutes.

5. Draw 0.5 µL from OPA vial (p/n 5061-3335).

6. Mix 4.0 µL in wash port 10 times default speed.

7. Draw 0.4 µL from FMOC vial (p/n 5061-3337).

8. Mix 4.4 µL in wash port 10 times default speed.

9. Draw 32 µL from injection diluent vial.

10.Mix 20 µL in wash port eight times.

11. Inject.

12.Wait 0.1 minutes.

13. Valve bypass

The location of the derivatization reagents and samples is upto the analyst and the ALS tray configuration. Using theG1367C with a 2 × 56 well plate tray (p/n G2258-44502), thelocations were:

• Vial 1: Borate buffer

• Vial 2: OPA

• Vial 3: FMOC

• Vial 4: Injection diluent

• P1-A-1: Sample

Thermostatted column compartment (TCC)Left and right temperatures were set to 40 °C. Enable analysiswhen the temperature is within ± 0.8 °C. See Table 5 forwhich heat sink to use.

Diode array detector (DAD)Signal A: 338 nm, 10 nm bandwidth, and reference

wavelength 390 nm, 20 nm bandwidth.

Signal B: 262 nm, 16 nm bandwidth, and reference wavelength 324 nm, 8 nm bandwidth.

Signal C: 338 nm, 10 nm bandwidth, and reference wavelength 390 nm, 20 nm bandwidth.

The DAD was programmed to switch to 262 nm, 16 nmbandwidth, reference wavelength 324 nm, 8 nm bandwidth,after lysine elutes, and before hydroxyproline elutes. Signal Cwas determined by examining signal A and B timeframesbetween peaks 20 and 21, then choosing a suitable point toswitch wavelengths. Once the switch time was establishedand programmed into the method, signals A and B wereoptional.

Peak width settings of > 0.01 minutes were used for allcolumns.

4

Results and Discussion

As can be seen in Figure 1, using the same chromatographicconditions, the separation was very similar. The elution orderof the mixture on both columns was the same, and as shownin Figure 2, the relationship of retention times of the aminoacid samples was highly correlated between an Eclipse PlusC18 and a Poroshell HPH-C18, with a correlation co-efficient

of 0.997. As can be seen in the chromatograms, the retentiontimes were slightly less on the Poroshell HPH-C18 column.Some chromatographic differences are notable. Thus,separation of leucine and lysine looks better on PoroshellHPH-C18, while the separation between lysine andhydroxyproline and the sarcosine/proline pair looks worse. Assuggested in previous application notes, the chromatographycan be altered to enhance resolution of desired peak pairs.

Figure 1. Comparison of an Agilent Poroshell HPH C18 to an Agilent ZORBAX Eclipse Plus C18 column using theAmino Acid Method.

Figure 2. Correlation of retention times using AgilentPoroshell HPH-C18 and Agilent ZORBAX Eclipse Plus C18columns.

min2 4 6 8 10 12

mAU A

B

0

10

20

30

40

50

60

min0

0

2 4 6 8 10 12

mAU

0

10

20

30

40

50

60

Agilent Poroshell HPH-C18,4.6 × 100 mm, 2.7 µm

Agilent ZORBAX Eclipse Plus C18,4.6 × 100 mm, 3.5 µm

1

5

4

32

11

109

8

7

6

12

19

1816

15

14

23

2221

20

1317

1

532

11

1097

6

12

1916

15

23

2221

1317

20

18

4

8 14

Peak ID1. Aspartic acid2. Glutamic acid3. Asparagine4. Serine 5. Glutamine 6. Histidine 7. Glycine 8. Threonine 9. Arginine 10. Alanine 11. Tyrosine 12. Cystine 13. Valine 14. Methionine15. Norvaline 16. Tryptophan 17. Phenylalanine 18. Isoleucine 19. Leucine 20. Lysine 21. Hydroxyproline 22. Sarcosine 23. Proline

0 2 4 6 8 10 12 140

2

4

5

6

10

12

14

Retention time (min) Agilent ZORBAX Eclipse Plus C18

y = 1.0201x – 0.5565R2 = 0.9977

Ret

entio

n tim

e (m

in)

Agi

lent

Por

oshe

ll H

PH-C

18

Column: Agilent Poroshell HPH C18, 4.6 × 100 mm, 2.7 µm (p/n 695975-702) or Agilent Eclipse Plus C18, 4.6 × 100 mm, 3.5 µm (p/n 959961-902)

Flow rate: 1.5 mL/min

Gradient: Time (min) %B0 20.35 213.4 5713.5 10015.7 10015.8 218 end

Table 1. Conditions for Figure 1.

5

Column dimensionsThe method can easily be scaled to different columndimensions. In this work, three column dimensions werestudied. All columns were 100 mm in length with 4.6, 3.0, or2.1 mm internal diameter, as shown in Figure 3. In this case,the only changes to the method were made by altering theflow rate. Table 1 lists the gradient program used throughout.Flow rates are changed geometrically with the diameter of thecolumn. The flow rate used with the 4.6 × 100 mm columnwas 1.5 mL/minute. The flow rates for the 3 and 2.1 mmcolumns were 0.62 and 0.21 mL/min, respectively. In allcases, the low-volume heat exchanger was used with shortred tubing to minimize extra column volume. Using theAgilent 1260 Infinity Binary LC with low dispersion heatingand tubing, the column pressure was approximately 175 bar.

We observed that retention time of all analytes increasedslightly (without changing selectivity) as columns werechanged from larger to smaller internal diameter. This is dueto the increase in gradient delay time. As the flow rates arescaled and consequently reduced from larger to smallercolumn ids, the gradient delay volume remains constant,thereby increasing the time it takes for the gradient to reachthe column. The difference in retention between variouscolumn ids could potentially be reduced or eliminated byscaling the gradient delay volume on the LC system (addingor removing capillary length/diameter/volume between thepump and column inlet).

Figure 3. Agilent Poroshell HPH-C18 100 mm columns of different inside dimension using the amino acid method.

B14139

A

B

C

min2 4 6 8 10 12

mAU

0

20

40

60

80

0.44

0 0.59

0

0.97

9 1.50

31.

666

2.75

12.

963

3.38

93.

551

3.80

13.

958

4.50

0

4.86

1

5.80

1

6.47

36.

612

6.85

26.

925

7.15

27.

256

7.41

5

7.69

9

8.08

2

8.38

48.

555

9.06

2

9.48

6

9.76

2

11.4

91

113

12

1110

9

8

7654

32

191817

16

15

14

23

2221

20

min2 4 6 8 10 12

mAU

020406080

100120

0.45

90.

599

1.02

9

1.60

21.

755

2.92

63.

139

3.57

83.

750 3.99

54.

153

4.37

9

4.71

3

5.06

6

6.01

1

6.68

46.

817

7.05

07.

119

7.35

57.

442

7.60

4

7.88

8

8.26

6

8.55

68.

727

9.24

0

9.66

4

9.95

0

11.6

65 11.8

58

12.1

72

min2 4 6 8 10 12

mAU

020406080

100120

1.09

2

1.70

1

3.28

63.

508 3.

999

4.18

4 4.43

44.

589

5.15

6

5.52

3

6.48

4

7.14

6 7.27

97.

514

7.61

37.

813

7.91

28.

081

8.36

1

8.63

28.

756

9.03

69.

197

9.71

4

10.1

4810

.400

12.1

19 12.3

08

12.6

22

4.6 mm id

3.0 mm id

2.1 mm id

B14139

B14139

6

Lot-to-lot variabilityBatch-to-batch or lot-to lot reproducibility is also an importfactor in method development. It is recommended that, beforea method is adopted, one of the earliest validation steps is toexamine the method performance on at least three columnsmade from different lots.

Following good validation practice, three columns loaded withparticles from different production batches were examined for4.6, 3.0, and 2.1 × 100 mm columns. The overlays of these

three sets are shown in Figures 4A-C. As can be seen inFigure 4A, the amino acid separation on the 4.6 × 100 mmcolumn achieved good peak as well as baseline separationshape for all compounds. No change in elution order wasnoted, and lot-to-lot reproducibility looked good. A slightchange in retention time can be seen in Figure 4A though thek’ remained constant. However, a slight change in thewavelength switch time is required as it is tied to the elutiontimes of lucine and hydroxyproline. Similar reproducibility isevident in Figures 4B and 4C for the smaller id columns.

A

B

C

B14139

B14138

B14141

min2 4 6 8 10 12

mAU

0

20

40

60

80

0.44

0 0.59

0

0.97

9 1.50

31.

666

2.75

12.

963

3.38

93.

551

3.80

13.

958

4.50

0

4.86

1

5.80

1

6.47

36.

612

6.85

26.

925

7.15

27.

256

7.41

57.

699

8.08

2

8.38

48.

555

9.06

2

9.48

6

9.76

2

11.4

9111

.689

12.0

79

min2 4 6 8 10 12

mAU

0

20

40

60

80

0.44

0 0.58

9

0.95

6

1.45

91.

603

2.68

42.

885

3.31

63.

478

3.71

43.

871

4.41

8

4.77

1

5.72

1

6.36

26.

514

6.79

46.

876

7.03

37.

157

7.31

7

7.59

9

7.98

7

8.28

38.

453

8.95

9

9.42

29.

624

11.3

3611

.491

11.8

61

min2 4 6 8 10 12

mAU

0

20

40

60

80

0.43

70.

572

0.92

5

1.41

41.

575

2.63

32.

828

3.25

93.

426

3.65

13.

808

4.02

2

4.37

7

4.70

2

5.65

7

6.30

86.

443

6.72

16.

793

7.08

07.

238

7.51

8

7.90

9

8.19

78.

365

8.86

8

9.32

7

9.56

8

10.5

57

11.2

7711

.442

11.7

86

113

12

1110

9

87

6543

2

19181716

15

14

23

22

21

20

ALS injector programming

Figure 4A. Separation of amino acid and internal standards on three lots of Agilent Poroshell HPH-C18, 4.6 × 100 mm, 2.7 µm (p/n 695975-702).

7

A

B

C

B14139

B14140

B14138

min2 4 6 8 10 12

mAU

020406080

100120

0.45

90.

599

1.02

9

1.60

21.

755

2.92

63.

139

3.57

83.

750 3.99

54.

153

4.37

9

4.71

3

5.06

6

6.01

1

6.68

46.

817

7.05

07.

119

7.35

57.

442

7.60

4

7.88

8

8.26

6

8.55

68.

727

9.24

0

9.66

4

9.95

0

11.6

65 11.8

58

12.1

72

min2 4 6 8 10 12

mAU

020406080

100120

0.57

6

0.92

1

1.39

61.

508

2.66

32.

841

3.30

93.

487 3.68

03.

847

4.15

7

4.48

5

4.80

9

5.86

1

6.53

16.

675

6.96

37.

042

7.23

77.

330

7.50

0

7.79

3

8.18

9

8.47

98.

650

9.17

0

9.60

5

9.89

0

10.2

22

10.8

91

11.4

9811

.608

11.7

92

12.1

16

min2 4 6 8 10 12

mAU

020406080

100120

0.45

40.

582

0.94

3

1.40

41.

517

2.68

32.

862 3.32

83.

502 3.69

93.

862

4.17

0

4.47

5

4.80

2

5.82

7

6.19

4

6.46

06.

617

6.70

46.

934

7.02

37.

144

7.26

77.

434

7.72

1

8.11

5

8.39

88.

568

9.08

09.

303

9.55

59.

753

10.7

08

11.4

5211

.578

11.7

9611

.979

1

13

12

1110

98

7

64

3

2

191817

15 232221

20

14 16

A

B

C

B14139

B14140

B14138

min2 4 6 8 10 12

mAU

020406080

100120

0.62

5

1.09

2

1.70

1

3.28

63.

508 3.

999

4.18

4 4.43

44.

589

4.85

4

5.15

6

5.52

3

6.48

4

7.14

6 7.27

97.

514

7.61

37.

813

7.91

28.

081

8.36

1

8.63

28.

756

9.03

69.

197

9.45

99.

714

9.88

1

10.1

4810

.400

11.9

312

.119 12

.308

12.6

22

min2 4 6 8 10 12

mAU

020406080

100120

0.62

0

1.07

3

1.65

1

3.22

13.

433 3.

923

4.10

74.

344

4.50

6

4.76

5

5.08

9

5.44

0

6.40

3

7.06

6 7.19

57.

440

7.53

37.

736

7.82

87.

993

8.27

4

8.66

4

8.94

19.

102

9.37

99.

611

9.79

510

.041

10.3

03

12.0

10 12.2

00

12.5

10

min2 4 6 8 10 12

mAU

020406080

100120

0.64

8

1.11

0

1.68

0

3.27

23.

479 3.

986

4.16

84.

406

4.55

3

4.84

3

5.14

7

5.49

3

6.46

2

7.10

3 7.22

6

7.50

37.

617

7.86

68.

043

8.31

3

8.72

0

8.98

59.

138

9.38

99.

644

10.1

0710

.310

12.0

10 12.1

62

12.5

02

113

12

1110

9

87

64

3

2 191817

15

23

2221

20

5

14

16

Figure 4B. Separation of amino acid and internal standards on three lots of Agilent Poroshell HPH-C18, 3 × 100 mm, 2.7 µm (p/n 695975-502).

Figure 4C. Separation of amino acid and internal standards on three lots of Agilent Poroshell HPH-C18, 2.1 × 100 mm, 2.7 µm (p/n 695775-702).

8

LifetimeColumn lifetime is an important consideration forchromatographers analyzing amino acid samples. Most silicacolumns lose efficiency after prolonged exposure to theseconditions. Kirkland et al. [9] and Tindall and Perry [10]discussed possible reasons for the reduced lifetime of silicacolumns in phosphate buffer, but both agree that columns donot last as long.

There are two approaches to achieving high pH stability insilica HPLC columns. One way is to employ special bondingchemistry, as in the Agilent ZORBAX Extend C18 column. Thiscolumn uses bidentate bonding to protect the silica fromdissolution at high pH. Another way to achieve high pHstability is to modify the silica itself, making it less soluble.The surface of Poroshell HPH particles are chemicallymodified to form an organic layer, resistant to silica dissolutionat high pH conditions, using a proprietary process [11].

Figure 5 is an overlay of four chromatograms. Single 4 Lbottles of mobile phase A and B were prepared. A single2.1 × 100 mm column was used for lifetime testing from aseries of 500 analyses over a period of four weeks. In thisseries, approximately 102 injections were made each weekusing freshly opened amino acid standard mix and reagents.At the end of the sequence, the column was flushed with100% B mobile phase for 40 minutes and the instrument wasshut down. In this manner, the method was run for 3.5 daysand the column was stored with no analysis for 3.5 days. Thissimulated typical practice in a lab where samples are run foran extended time, and then a column is washed and stored.Storing a column in 100% mobile phase B was recommendedin the original amino quant methods, and is common practicein many successful laboratories that frequently run aminoacids. A realistic lifetime study was carried out, showingexcellent lifetime of the column over one month of use, withover 500 standard injections, shutting down and storing aftereach sequence. As can be seen in Figure 5, the 17 amino acidsample lost no resolution and only a slight retention time shiftwas seen.

A

B

C

D

min0 2 4 6 8 10 12

min0 2 4 6 8 10 12

min0 2 4 6 8 10 12

min0 2 4 6 8 10 12

mAU

020406080

1.07

6

1.63

7

3.44

2

4.10

7

4.36

64.

532

4.79

3

5.12

3

5.48

6

6.45

2

7.50

27.

603 7.

884

8.05

1

9.00

89.

166

9.67

1

10.1

04

11.1

53

mAU

020406080

1.08

1

1.65

6

3.42

1

4.09

14.

342

4.50

3

4.76

3

5.10

5

5.44

8

6.41

7

7.47

2 7.56

8 7.84

28.

011

8.96

59.

116

9.62

2

10.0

70

11.1

20

mAU

020406080

1.06

1

1.58

9

3.40

3

4.07

34.

319

4.48

1

4.74

2

5.07

9

5.41

5

6.38

8

7.44

17.

539 7.80

77.

976

8.93

29.

084

9.58

6

10.0

41

11.0

83

mAU

020406080 1.

070

1.54

6

3.38

1

4.04

94.

290

4.45

1

4.71

5

5.06

0

5.38

5

6.36

4

7.42

0 7.50

9 7.77

17.

942

8.88

79.

039

9.53

3

9.97

1

11.0

23

Injection 25

Injection 225

Injection 400

Injection 500

Figure 5. Column lifetime test using an Agilent Poroshell HPH-C18, 2.1 × 100 mm column running an amino acid method.

9

Conclusions

Agilent Poroshell HPH-C18 has selectivity similar to totallyporous Agilent ZORBAX Eclipse Plus C18. This allows easytransfer of existing methods such as the amino acid method.In this work, no changes to the chromatographic conditionswere made although changes in the gradient could be done toimprove resolution on selected amino acids. In most cases,Poroshell HPH-C18 was slightly less retentive than totallyporous Eclipse Plus C18. The method was investigated with4.6, 3.0, and 2.1 mm × 100 mm columns. Use of the lowvolume column heater is recommended. In total, four particlelots were investigated, requiring only slight changes to thewavelength switch time. A realistic lifetime study was carriedout, showing excellent lifetime of the column over one monthof use, with over 500 standard injections, shutting down andstoring after each sequence.

References

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2. Schuster, R.; Apfel, A. A new technique for the analysis ofprimary and secondary amino acids; Application note,Hewlett-Packard Publication number 5954-6257, 1986.

3. Schuster, R. Determination of amino acids in biological,pharmaceutical, plant and food samples by automated precolumn derivatization and high-performance liquidchromatography. J. Chromatogr. B. 1988, 431, 271-284.

4. Henderson, Jr., J. W.; Ricker, R. D.; Bidlingmeyer, B. A.;Woodward, C. Rapid, Accurate, Sensitive and ReproducibleHPLC Analysis of Amino Acids; Application note, AgilentTechnologies, Inc. Publication number 5980-1193E, 2000.

5. Woodward, C.; Henderson, Jr., J. W.; Todd Wielgos, T.High-Speed Amino Acid Analysis (AAA) on Sub-TwoMicron Reversed-phase (RP) Columns; Application note,Agilent Technologies, Inc. Publication number5989-6297EN, 2007.

6. Gratzfeld-Huesgen, A. Sensitive and Reliable Amino AcidAnalysis in Protein Hydrolysates using the Agilent 1100Series HPLC; Application note, Agilent Technologies, Inc.Publication number 5968-5658EN, 1999.

7. Greene, J.; Henderson, Jr., J. W.; Wikswo, J. P. Rapid andPrecise Determination of Cellular Amino Acid Flux RatesUsing HPLC with Automated Derivatization withAbsorbance Detection; Application note, AgilentTechnologies, Inc. Publication number 5990-3283EN, 2009.

8. Henderson, Jr., J. W.; Brooks, A. Improved Amino AcidMethods using Agilent ZORBAX Eclipse Plus C18 Columnsfor a Variety of Agilent LC Instrumentation and SeparationGoals; Application note, Agilent Technologies, Inc.Publication number 5990-4547EN, 2010.

9. Kirkland, J. J.; van Straten, M. A.; Claessens, H. A.Reversed-phase high-performance liquid chromatographyof basic compounds at pH 11 with silica-based columnpackings. J. Chromatogr. A. 1998, 797, 111-120.

10. Tindall, G. W.; Perry, R. L. Explanation for the enhanceddissolution of silica column packing in high pH phosphateand carbonate buffers. J. Chromatogr. A. 2003, 988,309-312.

11. Anon. Extending Column Lifetime in PharmaceuticalMethods with High pH-Stable Poroshell HPH Chemistries;Technical overview, Agilent Technologies, Inc. Publicationnumber 5991-5022EN, 2014.

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